Cytochrome c acetylation

ABSTRACT

Modulation of cytochrome c acetylation, e.g., with a SIR polypeptide, enables interventions that modulate lifespan regulation and cell proliferation, e.g., by modulating apoptosis and/or mitochondrial function such as respiration.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Application Ser. No.60/433,096, filed on Dec. 13, 2002, the contents of which areincorporated by reference herein for all purposes.

BACKGROUND OF THE INVENTION

Cytochrome c has been identified as an important participant inapoptosis. In living cells, cytochrome c is present in the intermembranespace of the mitochondria, where it plays a role in respiration. Duringapoptosis, however, cytochrome c translocates to the cytosol. Green etal. (1998) Cell 94:695-698; Martinou et al. (1999) Nature 399:411-412.Mitochondrial-produced reactive oxygen species (ROS) occur in bursts inthe mitochondria and are involved in release of cytochrome c into thecytosol. Kirkland (2001) J. Neurosci 21(6):1949-1963. In the cytosol,cytochrome c binds to Apaf-1 in a dATP/ATP dependent manner,precipitating the oligomerization of Apaf-1. Kluck et al. (2000) J.Biol. Chem. 21:16127-16133. The ensuing recruitment and activation ofcaspase-9 results in the activation of further caspases, includingcaspase-3. These caspases in turn cleave many important substrates andorchestrate the final packaging of the apoptotic cell. Liu et al. (1997)Cell 89:175-184; Enari et al. (1998) Nature 391:43-50; Sahara (199)Nature 401:168-173.

SUMMARY OF THE INVENTION

The invention is based, in part, on the discovery that by modulatingacetylation of cytochrome c, cytochrome c induced cellular processessuch as apoptosis and mitochondrial function (e.g., respiration) can bemodulated. For example, it has been found that proteins of the SilentInformation Regulator (SIR) family, such as SIRT1, SIRT2, SIRT3, SIRT4,SIRT5, SIRT6 and SIRT7, interact with cytochrome c and reduceacetylation of cytochrome c, thereby affecting apoptosis and/ormitochondrial function. Accordingly, the invention relates to methodsand compositions employing a polypeptide (or polypeptides) having theability to modulate acetylation (e.g., a SIR polypeptide orpolypeptides) and agonists or antagonists thereof to modulate cytochromec related processes such as apoptosis or mitochondrial function.

In one aspect, the invention features a method of screening a compound,e.g., for its effect on the acetylation status of a cytochrome cpolypeptide. The method includes providing a compound, e.g., a compoundwhich interacts with (e.g., binds to) cytochrome c; contacting acytochrome c polypeptide with the compound and evaluating a chemicalreaction, e.g., the effect of the compound on the acetylation of thecytochrome c polypeptide.

In a preferred embodiment, the cytochrome c polypeptide is acetylatedand the ability of the compound to deacetylate the cytochrome cpolypeptide is evaluated. The acetylated cytochrome c polypeptide caninclude a moiety that can be spectroscopically detected, e.g., afluorophore or a chromophore. Cytochrome c can also be evaluated, e.g.,using mass spectroscopy (e.g., MALDI-TOF), or an immunoassay, e.g.,using an antibody (e.g., an antibody specific for an acetylated form) orother specific binding agent.

The method can include, e.g., evaluating the cytochrome c polypeptidedirectly, evaluating a modification product of the cytochrome cpolypeptide (e.g., by contacting a reaction component to a developer,e.g., a protease), or evaluating a reaction substrate (e.g., thecytochrome c polypeptide or NAD) or byproduct (e.g., nicotinamide orO-acetyl ADP ribose) or parameter (e.g., temperature, pH).

The cytochrome c polypeptide can be a full length cytochrome c or afragment thereof, e.g., a peptide of at least 3, 4, 5, or more aminoacids, e.g., between 3-12 or 4-10 amino acids. The cytochrome cpolypeptide can be from a mammalian cytochrome c, e.g., a human, rat,mouse, canine, ovine, or bovine cytochrome c.

In one embodiment, the compound is other than a SIR protein, or otherthan a protein.

In one embodiment, the method includes providing a SIR protein inaddition to the compound.

In one embodiment, the compound is a SIR protein, e.g., a wildtype ormutant SIR protein

In some embodiments, the interaction between the compound and cytochromec polypeptide is evaluated in vitro, e.g., using an isolatedpolypeptide. The cytochrome c polypeptide can be in solution (e.g., in amicelle) or bound to a solid support, e.g., a column, agarose beads, aplastic well or dish, or a chip (e.g., a microarray). Similarly, thecompound can be in solution or bound to a solid support.

In other embodiments, interaction of a compound with the cytochrome cpolypeptide and/or the ability of the compound to effect the acetylationstatus of the cytochrome c polypeptide can be evaluated using acell-based assay. For example, the cell can be a yeast cell, aninvertebrate cell (e.g., a fly cell), or a vertebrate cell (e.g., aXenopus oocyte or a mammalian cell, e.g., a mouse or human cell).

In preferred embodiments, the cell-based assay measures the acetylationor deacetylation of the cytochrome c polypeptide and/or measures thebinding of cytochrome c to a second protein (e.g., Apaf-1 or a fragmentthereof) and/or measures cell viability and apoptosis, to evaluate theacetylation status of cytochrome c. In other embodiments, a compoundidentified as altering the acetylation status of cytochrome c can beevaluated for its effect on mitochondrial function such as respiration.

Possible test compounds include, e.g., small organic and inorganicmolecules, peptides, antibodies, and nucleic acid molecules.

In one embodiment, a co-factor for deacetylation activity, e.g., a SIRpolypeptide co-factor, such as NAD or an NAD analog, is also present.

In some embodiments, one or more steps of the method are repeated one ormore times such that, e.g., a library of test compounds can beevaluated.

In another aspect, the invention features a method of screening acompound, e.g., a compound which modulates, e.g., increases ordecreases, interaction between a polypeptide which modulatesacetylation, e.g., a polypeptide having deacetylation activity, andcytochrome c. The method includes: contacting a polypeptide havingacetylation or deacetylation activity or fragment thereof, with acompound, e.g., a modulator of the polypeptide, in the presence ofcytochrome c or a fragment thereof, and determining if the compoundmodulates interaction, e.g., binding, between the polypeptide and thecytochrome c. In one embodiment, the polypeptide has deacetylationactivity and is, e.g., a SIR polypeptide. Preferably, the SIRpolypeptide is, e.g., a SIRT1 polypeptide, a SIRT2 polypeptide, a SIRT3polypeptide, a SIRT4 polypeptide, a SIRT5 polypeptide, a SIRT6polypeptide, a SIRT7 polypeptide, or combinations thereof. In someembodiments, the ability of the compound to modulate interaction, e.g.,binding, between the polypeptide and cytochrome c can be determined byevaluating the interaction, e.g., binding, of the polypeptide orfragment thereof, and the cytochrome c or fragment thereof in theabsence of the compound. Possible compounds include, e.g., small organicor inorganic molecules, peptides, antibodies, and nucleic acidmolecules.

In one embodiment, a co-factor for deacetylation activity, e.g., a SIRpolypeptide co-factor, such as NAD or an NAD analog, is also presentduring the contacting step.

In one embodiment, the cytochrome c or a fragment thereof, is acetylatedor labeled.

In some embodiments, the method further includes determining if thecompound modulates acetylation of cytochrome c. For example, acetylationof the cytochrome c or fragment thereof can be evaluated in the absenceand presence of the compound to determine the effect the compound has onthe polypeptide acetylation or deacetylation activity.

In some embodiments, the method is repeated one or more times such that,e.g., a library of test compounds can be evaluated.

In some embodiments, the method can further include evaluating acompound that modulates interaction between a polypeptide havingacetylation or deacetylation activity, or a fragment thereof, andcytochrome c, or fragment thereof, to determine its effect on aparameter of a cell, for example, an age-associated parameter of a cell(e.g., a fibroblast, an osteoblast, a skin cell, a blood cell, atransformed cell, a senescent cell, a cultured cell or a neural cell),e.g., by contacting the cell with the compound. In such embodiments,modulation of the interaction of the polypeptide and a cytochrome c andmodulation of an age-associated parameter relative to a control cellidentifies the compound as having a modulatory effect on life spanregulation. Such compounds can, e.g., be identified as a candidate formodulating, e.g., slowing or speeding, age, modulating, e.g., increasingor decreasing lifespan, modulating, e.g., increasing or decreasingmetabolism (e.g., by increasing or decreasing metabolic function and/orrate), modulating, e.g., increasing or decreasing electron transport.The age associated parameter can be, e.g., one or more of: (i) lifespanof the cell; (ii) presence or abundance of a gene transcript or geneproduct in a cell or organism that has a biological age dependentexpression pattern; (iii) resistance of the cell or organism to stress;(iv) one or more metabolic parameters of the cell or organism; (v)proliferative capacity of the cell or a set of cells present in theorganism. In some embodiments, the parameter is one or more of (i) theproliferative capacity of the cell or a set of cells in an organism and(ii) apoptosis of the cell or a set of cells in the organism, and, e.g.,compounds which decrease the proliferative capacity can be identified ascandidate compounds for treating or preventing a disorder associatedwith unwanted cell proliferation, e.g., cancer.

In another aspect, the invention features a method of screening acompound, e.g., a compound which modulates acetylation or deacetylationactivity of a polypeptide. The method includes: contacting a cell whichexpresses a polypeptide having acetylation or deacetylation activity andcytochrome c (or fragments thereof) with a compound, and determining ifthe compound modulates, e.g., increases or decreases, acetylation ofcytochrome c. In one embodiment, the polypeptide has deacetylationactivity and is, e.g., a SIR polypeptide. Preferably, the SIRpolypeptide is, e.g., a SIRT1 polypeptide, a SIRT2 polypeptide, a SIRT3polypeptide, a SIRT4 polypeptide, a SIRT5 polypeptide, a SIRT6polypeptide, a SIRT7 polypeptide, or combinations thereof. Compoundswhich increase acetylation of cytochrome c can be identified ascompounds which inhibit or reduce a SIR polypeptide activity, i.e.,deacetylation. Compounds which modulate acetylation of cytochrome c canbe identified as compounds which modulate acetylation or deacetylationactivity of the polypeptide. Possible compounds include, e.g., smallorganic or inorganic molecules, peptides, antibodies, and nucleic acidmolecules.

In some embodiments, the ability of a compound to modulate acetylationcan be evaluated by determining increases or decreases in theinteraction, e.g., binding, of cytochrome c and a secondary protein(e.g., an Apaf-1 protein or fragment thereof). The ability of a compoundto modulate the acetylation or deacetylation activity of thepolypeptide, e.g., increase or decrease polypeptide deacetylationactivity, can be accomplished by comparing the interaction, e.g.,binding, of cytochrome c and Apaf-1 in the cell in the absence andpresence of the compound. Compounds which increase the interactionbetween cytochrome c and Apaf-1 can be identified as compounds whichdecrease acetylation and/or increase deacetylation of cytochrome c.Compounds which decrease the interaction can be identified as compoundswhich increase acetylation and/or decrease deacetylation of cytochromec. In other embodiments, the ability of a compound to modulateacetylation can be identified by evaluating acetylation, e.g., in theabsence and presence of the compound.

In one embodiment, a co-factor for deacetylation activity, e.g., a SIRpolypeptide co-factor, such as NAD or a NAD analog, is also present.

In one embodiment, the cytochrome c or a fragment thereof, is acetylatedor labeled.

In some embodiments, the method is repeated one or more times such that,e.g., a library of test compounds can be evaluated.

In some embodiments, the method can further include evaluating acompound that modulates the acetylation or deacetylation activity of thepolypeptide, to determine its effect on a parameter of a cell, forexample, an age-associated parameter of a cell (e.g., a fibroblast, anosteoblast, a skin cell, a blood cell, a transformed cell, a senescentcell, a cultured cell or a neural cell). The cell can be the same cellused to evaluate the ability of the compound to modulate thepolypeptide's acetylation or deacetylation capacity, or it can be adifferent cell which is contacted with the compound. In suchembodiments, modulation of the polypeptide's acetylation ordeacetylation capacity and modulation of an age-associated parameterrelative to a control cell identify the compound as having a modulatoryeffect on life span regulation. Such compounds can be identified ascandidates for modulating, e.g., slowing or speeding, age, modulating,e.g., increasing or decreasing lifespan, modulating, e.g., increasing ordecreasing metabolism (e.g., by increasing or decreasing metabolicfunction and/or rate), modulating, e.g., increasing or decreasing,electron transport. The age-associated parameter can be, e.g., one ormore of: (i) lifespan of the cell; (ii) presence or abundance of a genetranscript or gene product in a cell or organism that has a biologicalage dependent expression pattern; (iii) resistance of the cell ororganism to stress; (iv) one or more metabolic parameters of the cell ororganism; (v) proliferative capacity of the cell or a set of cellspresent in the organism. In some embodiments, the parameter is one ormore of (i) the proliferative capacity of the cell or a set of cells inan organism and (ii) apoptosis of the cell or a set of cells in theorganism, and, e.g., compounds which decrease the proliferative capacitycan be identified as candidate compounds for treating or preventing adisorder associated with unwanted cell proliferation, e.g., cancer.

In another aspect, the invention features a method of screening acompound which includes: providing a compound which interacts withcytochrome c or a polypeptide having acetylation or deacetylationactivity, e.g., a compound that binds the polypeptide or cytochrome c;contacting a cell or organism that expresses the polypeptide orcytochrome c with the compound, and evaluating the effect of thecompound on acetylation or deacetylation of cytochrome c. In oneembodiment, the polypeptide has deacetylation activity and is, e.g., aSIR polypeptide. Preferably, the SIR polypeptide is, e.g., a SIRT1polypeptide, a SIRT2 polypeptide, a SIRT3 polypeptide, a SIRT4polypeptide, a SIRT5 polypeptide, a SIRT6 polypeptide, a SIRT7polypeptide, or combinations thereof.

In other embodiments, the method further includes evaluating a cell ororganism to determine the effect of the compound on a parameter of acell, for example, an age-associated parameter of a cell (e.g., afibroblast, an osteoblast, a skin cell, a blood cell, a transformedcell, a senescent cell, a cultured cell or a neural cell). The cell canbe the same cell used to evaluate the ability of the compound tomodulate the polypeptide's acetylation or deacetylation capacity, or itcan be a different cell which is contacted with the compound. In suchembodiments, interaction with the polypeptide or cytochrome c andmodulation of an age-associated parameter relative to a control cellidentifies the compound as having a modulatory effect on life spanregulation. Such compounds can be identified as candidates formodulating, e.g., slowing or speeding, age, modulating, e.g., increasingor decreasing lifespan, modulating, e.g., increasing or decreasingmetabolism (e.g., by increasing or decreasing metabolic function and/orrate), modulating, e.g., increasing or decreasing electron transport.The age-associated parameter can be, e.g., one or more of: (i) lifespanof the cell; (ii) presence or abundance of a gene transcript or geneproduct in a cell or organism that has a biological age dependentexpression pattern; (iii) resistance of the cell or organism to stress,e.g., genotoxic stress (e.g., etopicide, UV irradiation, exposure to amutagen, and so forth) or oxidative stress or hypoxic stress; (iv) oneor more metabolic parameters of the cell or organism (e.g., proteinsynthesis or degradation, ubiquinone biosynthesis, cholesterolbiosynthesis, ATP levels, glucose metabolism, nucleic acid metabolism,ribosomal translation rates, etc.); (v) proliferative capacity of thecell or a set of cells present in the organism (e.g., of retinal cells,bone cells, white blood cells, etc.). Alternatively, evaluating the rateof aging can include directly measuring the average life span of a groupof animals (e.g., a group of genetically matched animals) and comparingthe resulting average to the average life span of a control group ofanimals (e.g., a group of animals that did not receive the compound butare genetically matched to the group of animals that did receive thetest compound). In other embodiments, the parameter is one or more of(i) the proliferative capacity of the cell or a set of cells in anorganism and (ii) apoptosis of the cell or a set of cells in theorganism, and, e.g., compounds which decrease the proliferative capacitycan be identified as candidate compounds for treating or preventing adisorder associated with unwanted cell proliferation, e.g., cancer.

In some embodiments, the cell is a transgenic cell, e.g., a cell havinga transgene. In some embodiments, the transgene encodes a protein thatis normally exogenous to the transgenic cell. In some embodiments, thetransgene encodes a human protein, e.g., a human polypeptide havingacetylation or deacetylation activity (e.g., a SIR polypeptide) or ahuman cytochrome c polypeptide. In some embodiments, the transgene islinked to a heterologous promoter. In other embodiments, the transgeneis linked to its native promoter. In some embodiments, the cell isisolated from an organism that has been contacted with the compound. Inother embodiments, the cell is contacted directly with the compound.

In some embodiments, the organism is on a calorically rich diet, whilein other embodiments the organism is on a calorically restricted diet.

In some embodiments, a portion of the organism's life, e.g., at least20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, of the expected lifespan of the organism, has elapsed prior to the organism being contactedwith the compound.

In some embodiments, the method can further include evaluating theability of the compound to interact, e.g., bind to a polypeptide havingacetylation or deacetylation activity or a cytochrome c polypeptide,e.g., evaluating the interaction prior to evaluating the effect of thecompound on a cell or organism. In some embodiments, the interactionbetween the test compound and the polypeptide or cytochrome c isevaluated in vitro, e.g., using an isolated polypeptide. The polypeptideor cytochrome c polypeptide can be in solution (e.g., in a micelle) orbound to a solid support, e.g., a column, agarose beads, a plastic wellor dish, or a chip (e.g., a microarray). Similarly, the test compoundcan be in solution or bound to a solid support.

In other embodiments, when the method includes evaluating the ability ofthe compound to interact, e.g., bind to, the polypeptide or cytochromec, the interaction between the compound and the polypeptide orcytochrome c is evaluated using a cell-based assay. For example, thecell can be a yeast cell, an invertebrate cell (e.g., a fly cell), or avertebrate cell (e.g., a Xenopus oocyte or a mammalian cell, e.g., amouse or human cell). In preferred embodiments, the cell-based assaymeasures the activity or expression levels of the polypeptide orcytochrome c.

Possible test compounds include, e.g., small organic or inorganicmolecules, peptides, antibodies, and nucleic acid molecules.

In one embodiment, a co-factor for deacetylation activity, e.g., a SIRpolypeptide co-factor, such as NAD or an NAD analog, is also present.

In one embodiment, the cytochrome c or a fragment thereof, is acetylatedor labeled.

In some embodiments, one or more steps of the method are repeated one ormore times such that, e.g., a library of test compounds can beevaluated.

In another aspect, the invention features a method of evaluating aprotein, comprising: identifying or selecting a candidate protein,wherein the candidate protein is a polypeptide having acetylation ordeacetylation activity, or a cytochrome c polypeptide; altering thesequence, expression or activity of the candidate protein in a cell orin one or more cells of an organism; and determining whether thealteration has an effect on the interaction, e.g., binding, of thepolypeptide with a cytochrome c polypeptide, or on the deacetylation ofcytochrome c. In one embodiment, the polypeptide has deacetylationactivity and is, e.g., a SIR polypeptide. Preferably, the SIRpolypeptide is, e.g., a SIRT1 polypeptide, a SIRT2 polypeptide, a SIRT3polypeptide, a SIRT4 polypeptide, a SIRT5 polypeptide, a SIRT6polypeptide, a SIRT7 polypeptide, or combinations thereof.

In some embodiments, the candidate protein is identified byamplification of the gene or a portion thereof encoding the candidateprotein, e.g., using a method described herein, e.g., PCR amplificationor the screening of a nucleic acid library. In preferred embodiments,the candidate protein is identified by searching a database, e.g.,searching a sequence database for protein sequences homologous to thepolypeptide or cytochrome c.

In preferred embodiments, the candidate protein is a human protein. Inother embodiments, the candidate protein is a mammalian protein, e.g., amouse protein. In other embodiments, the protein is a vertebrateprotein, e.g., a fish, bird or reptile protein, or an invertebrateprotein, e.g., a worm or insect protein. In still other embodiments, theprotein is a eukaryotic protein, e.g., yeast protein.

In another aspect, the invention features method of evaluating aprotein, the method includes: a) identifying or selecting a candidateprotein, wherein the candidate protein is a polypeptide havingacetylation or deacetylation activity, or a cytochrome c polypeptide; b)identifying one or more polymorphisms in a gene, e.g., one or more SNPsthat encodes the candidate protein; and c) assessing correspondencebetween the presence of one or more of the polymorphisms and aninteraction, e.g., binding, of the polypeptide with the cytochrome c, orwith the deacetylation of the cytochrome c. In one embodiment, thepolypeptide has deacetylation activity and is, e.g., a SIR polypeptide.Preferably, the SIR polypeptide is, e.g., a SIRT1 polypeptide, a SIRT2polypeptide, a SIRT3 polypeptide, a SIRT4 polypeptide, a SIRT5polypeptide, a SIRT6 polypeptide, a SIRT7 polypeptide, or combinationsthereof. The polymorphisms can be naturally occurring or laboratoryinduced. In one embodiment, the organism is an invertebrate, e.g., a flyor nematode; in another embodiment the organism is a mammal, e.g., arodent or human. A variety of statistical and genetic methods can beused to assess correspondence between a polymorphism and longevity. Suchcorrelative methods include determination of linkage disequilibrium, LODscores, and the like.

In another aspect, the invention features a method of screening for acompound, e.g., a compound that modulates a polypeptide havingacetylation or deacetylation activity to identify agonists orantagonists of the polypeptide. The method includes providing a cellwhich expresses cytochrome c and which either over- or under-expressesthe polypeptide, contacting the cell with a compound; and evaluating thecompound for its ability to modulate acetylation in the cell.Acetylation status of cytochrome c can be evaluated and/or cellviability and apoptosis can be evaluated. In some embodiments, thecompound can further by evaluated for its effect on mitochondrialfunction, e.g., respiration.

In some embodiments, when screening for a deacetylation antagonist, thecell can be a cell which over-expresses a polypeptide havingdeacetylation activity, and the effect of the compound acetylationstatus of cytochrome c can be evaluated. Possible compounds include,e.g., small organic or inorganic molecules, peptides, antibodies, andnucleic acid molecules. The compound can be evaluated by determiningacetylation status of cytochrome c and/or determining cell viability andapoptosis. For example, an antagonist can be selected which reducesprogrammed cell death and apoptosis of the cell and/or which decreasedeacetylation and/or increase acetylation of cytochrome c in the cell.The cell can be, e.g., a cell which includes a sequence encoding thepolypeptide under the control of a regulatory sequence, e.g., aregulatory sequence which does not naturally control expression of thepolypeptide, e.g., an inducible regulatory sequence, which results inincreased levels of the polypeptide being expressed in the cell. In someembodiments, the method is repeated one or more times such that, e.g., alibrary of test compounds can be evaluated. Such antagonists may beuseful, e.g., as candidate compounds for modulating, e.g., decreasing,the rate of aging. These candidate compounds can be evaluated, e.g., byany of the methods described herein to evaluate the rate of aging.

In other embodiments, when screening for a deacetylation agonist, thecell can be a cell which under-expresses a polypeptide havingdeacetylation activity, and the effect on acetylation status ofcytochrome c can be evaluated. Possible compounds include, e.g., smallorganic and inorganic molecules, peptides, antibodies, and nucleic acidmolecules. The compound can be evaluated by determining acetylationstatus of cytochrome c and/or determining cell viability and apoptosis.For example, an agonist can be selected which increases or inducesprogrammed cell death and apoptosis of the cell and/or which increasedeacetylation and/or decrease acetylation in the cell. The cell can be,e.g., a cell which includes a sequence encoding the polypeptide underthe control of a regulatory sequence, e.g., a regulatory sequence whichdoes not naturally control expression of the polypeptide, e.g., aninducible regulatory sequence, which results in decreased levels of thepolypeptide being expressed in the cell. In other embodiments, the cellscan under-express the polypeptide due to an agent which decreasesexpression of the polypeptide, e.g., antisense, and/or RNAi. In someembodiments, the method is repeated one or more times such that, e.g., alibrary of test compounds can be evaluated. Such agonists may be useful,e.g., as candidate compounds for aging related disorders and senescencerelated disorders. These agonist may also be useful, e.g., ascandidates, for treating or preventing unwanted cell growth, e.g.,cancer, inflammatory and autoimmune disorders, Alzheimer's disease.These candidate compounds can be evaluated, e.g., by any of the methodsdescribed herein to evaluate the rate of aging or effect on unwantedcell proliferation.

In one embodiment, the polypeptide has deacetylation activity and is,e.g., a SIR polypeptide. Preferably, the SIR polypeptide is, e.g., aSIRT1 polypeptide, a SIRT2 polypeptide, a SIRT3 polypeptide, a SIRT4polypeptide, a SIRT5 polypeptide, a SIRT6 polypeptide, a SIRT7polypeptide, or combinations thereof. In some embodiments, whenscreening for a SIR antagonist, the cell can be a cell whichover-expresses a SIR polypeptide, and the effect of the compound on SIRexpression and/or activity can be evaluated. Possible compounds include,e.g., small organic or non-organic molecules, peptides, antibodies, andnucleic acid molecules. In one embodiment, the expression levels of theSIR polypeptide prior to and after administration of a compound to thecell can be evaluated, and compounds which decrease the expression levelof the SIR polypeptide can be selected as SIR antagonists. A decrease inthe expression level is any statistically significant decrease in a SIRpolypeptide expression levels. In some preferred embodiments, SIRantagonist are selected which result in SIR expression levels in theover-expressing cell to return to levels comparable to the same celltype but which has not been modified to increase SIR levels. In otherembodiments, the cell can be a cell which over-expresses a SIRpolypeptide, and compounds which decrease SIR expression and effect anactivity can be evaluated for their effect on a SIR activity, e.g.,apoptosis and/or deacetylation. For example, SIR antagonist can beselected which reduces programmed cell death and apoptosis of the celland/or which decrease deacetylation and/or increase acetylation in thecell. The cell can be, e.g., a cell which includes a SIR encodingsequence under the control of a regulatory sequence, e.g., a non-SIRregulatory sequence, e.g., an inducible regulatory sequence, whichresults in increased levels of SIR being expressed in the cell. Suchantagonists may be useful, e.g., as candidate compounds for modulating,e.g., decreasing, the rate of aging. These candidate compounds can beevaluated, e.g., by any of the methods described herein to evaluate therate of aging.

In other embodiments, when screening for a SIR agonist, the cell can bea cell which under-expresses a SIR polypeptide, and the effect of thecompound on SIR expression and/or activity can be evaluated. Possiblecompounds include, e.g., small organic or inorganic molecules, peptides,antibodies, and nucleic acid molecules. In one embodiment, theexpression levels of SIR prior to and after administration of a compoundto the cell can be evaluated, and compounds which increase theexpression level of SIR can be selected as SIR agonists. An increase inthe expression level is any statistically significant increase in SIRexpression levels. In some preferred embodiments, SIR agonist areselected which result in SIR expression levels in the under expressingcell to return to levels comparable to the same, cell type but which hasnot been modified to reduce SIR levels. In other embodiments, the cellcan be a cell which under-expresses SIR, and compounds which increaseSIR expression and activity can be evaluated for their effect on a SIRactivity, e.g., apoptosis and/or deacetylation. For example, SIR agonistcan be selected which increases or induces programmed cell death andapoptosis of the cell and/or which increase deacetylation and/ordecrease acetylation in the cell. The cell can be, e.g., a cell whichincludes a SIR encoding sequence under the control of a regulatorysequence, e.g., a non-SIR regulatory sequence, e.g., an inducibleregulatory sequence, which results in decreased levels of SIR beingexpressed in the cell. In other embodiments, the cells can under-expressSIR due to an agent which decreases SIR expression, e.g., a SIRantisense, RNAi. In some embodiments, the method is repeated one or moretimes such that, e.g., a library of test compounds can be evaluated.Such agonists may be useful, e.g., as candidate compounds for agingrelated disorders and senescence related disorders. These agonist mayalso be useful, e.g., as candidates, for treating or preventing unwantedcell growth, e.g., cancer, inflammatory and autoimmune disorders,Alzheimer's disease. These candidate compounds can be evaluated, e.g.,by any of the methods described herein to evaluate the rate of aging oreffect on unwanted cell proliferation.

In another aspect, the invention features a method of modulating cellgrowth in an animal, e.g., a mammal, by modulating the acetylationstatus of a cytochrome c in the animal. In some embodiments, cell growthcan be modulated using an antagonist or agonist of deacetylation. Forexample, in some embodiments, an antagonist or agonist of a SIRpolypeptide, e.g., a SIRT1 polypeptide, a SIRT2 polypeptide, a SIRT3polypeptide, a SIRT4 polypeptide, a SIRT5 polypeptide, a SIRT6polypeptide, a SIRT7 polypeptide, can be used.

In one embodiment, the method includes modulating cell growth byincreasing acetylation of cytochrome c. An increase in acetylation ofcytochrome c can reduce or inhibit apoptosis of a cell. In a furtherembodiment, the method includes inactivating a polypeptide havingdeacetylation activity, e.g., by the use of antisense, RNAi, antibodies,intrabodies, NAD depletion, a dominant negative mutant of thepolypeptide, or by the addition of cofactor-analogs, e.g., NAD analogssuch as those described in Vaziri et al. (1997) or nicotinamide. In afurther embodiment, the method includes introducing adeacetylation-resistant form of cytochrome c.

In another embodiment, the method includes modulating cell growth bydecreasing acetylation of cytochrome c. A decrease in acetylation ofcytochrome c can increase or induce apoptosis in a cell. In a furtherembodiment, the method includes increasing NAD concentrations. In afurther embodiment, the method includes increasing concentrations of apolypeptide having deacetylation activity, e.g. by addition of purifiedpolypeptide, by expression of the polypeptide from heterologous genes,or by increasing the expression of endogenous polypeptide, or by theaddition of cofactor-analogs, e.g., NAD analogs such as those describedin Vaziri et al. (1997). In still another embodiment, the invention is amethod for treating a mammal, e.g., a mammal having a diseasecharacterized by unwanted cell proliferation, e.g., cancer, acceleratedsenescence-related disorders, inflammatory and autoimmune disorders,Alzheimer's disease, and aging-related disorders, e.g., a human mammal.The method can also be used to treat a disorder described in Ser. No.10/656,530 or 60/488,261.

The present invention also relates to a method of modulating the growthof a cell in vivo or in vitro by modulating the deacetylation of acytochrome c in the cell.

In one embodiment, the method includes modulating the growth of a cellby increasing acetylation of cytochrome c, thereby increasing cellgrowth. In a further embodiment, the method includes inactivating apolypeptide having deacetylation activity, e.g., by the use ofantisense, RNAi, antibodies, intrabodies, NAD depletion, a dominantnegative mutant of the polypeptide, or nicotinamide, or decreasing thepolypeptide's activity by the addition of cofactor-analogs, e.g., NADanalogs such as those described in Vaziri et al. (1997). In a furtherembodiment, the method includes introducing a deacetylation-resistantform of cytochrome c. In one embodiment, the polypeptide is a SIRpolypeptide. Preferably, the SIR polypeptide is, e.g., a SIRT1polypeptide, a SIRT2 polypeptide, a SIRT3 polypeptide, a SIRT4polypeptide, a SIRT5 polypeptide, a SIRT6 polypeptide, a SIRT7polypeptide, or combinations thereof.

In one embodiment, the method includes modulating the growth of a cellby decreasing acetylation of cytochrome c, thereby affecting cellgrowth. In a further embodiment, the method includes increasing NADconcentrations. In a further embodiment, the method includes increasingconcentrations of a polypeptide having deacetylation activity, e.g. byaddition of purified polypeptide, by expression of the polypeptide fromheterologous genes, or by increasing the expression of endogenoussequence encoding the polypeptide, or by the addition ofcofactor-analogs, e.g., NAD analogs such as those described in Vaziri etal. (1997) and U.S. 2003/0207325 (Ser. No. 09/461,580).

In another aspect, the invention is a method for treating or preventinga disease characterized by unwanted cell proliferation, e.g., cancer, ina subject. The method includes administering a deacetylation agonist.For example, the agonist can be one or more of: a purified polypeptidehaving deacetylation activity, by expression of such a polypeptide fromheterologous genes, or by increasing the expression of an endogenoussequence encoding such a polypeptide, and other compounds identified bya method described herein, e.g., compounds that induce apoptosis in acell, e.g., a cell expressing a polypeptide having deacetylationactivity. In one embodiment, the polypeptide a SIR polypeptide.Preferably, the SIR polypeptide is, e.g., a SIRT1 polypeptide, a SIRT2polypeptide, a SIRT3 polypeptide, a SIRT4 polypeptide, a SIRT5polypeptide, a SIRT6 polypeptide, a SIRT7 polypeptide, or combinationsthereof.

In a preferred embodiment, the method includes administering adeacetylation agonist in combination with one or more therapeuticagents, e.g., a therapeutic agent or agent for treating unwanted cellproliferation. The therapeutic agents include, for example, one or moreof a chemotherapeutic agent, a radioisotope, and a cytotoxin. Examplesof chemotherapeutic agents include taxol, cytochalasin B, gramicidin D,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,busulfan, cisplatin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, chlorambucil, gemcitabine,actinomycin, procaine, tetracaine, lidocaine, propranolol, puromycin,maytansinoids and analogs or homologs thereof. Additional therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, CC-1065, melphalan, carinustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine, vinblastine, taxol and maytansinoids). Radioisotopescan include alpha, beta and/or gamma emitters. Examples of radioisotopesinclude ²¹²Bi ²¹³Bi, ¹³¹I, ²¹¹At, ¹⁸⁶Re, ⁹⁰Y and ¹¹⁷Lu.

The agonist and the therapeutic agents can be administeredsimultaneously or sequentially.

The subject can be a human subject or a non-human subject, e.g., ananimal model.

In another aspect, the invention features a method of treating orpreventing a disease or disorder. The method includes modulatingcytochrome c deacetylation activity of a SIR polypeptide in a subject,e.g., by providing a modulator of SIR cytochrome c deacetylationactivity to the subject. The modulator can be provided by administeringthe modulator to the subject, or, e.g., by an ex vivo method in whichcells of the subject or of another organism are contacted with themodulator and then administered to the subject. Exemplary modulatorsinclude compounds that alter binding or access to an active site of aSIR polypeptide (e.g., an antibody or NAD analog, e.g., nicotinamide),compounds that alter the expression level of a SIR polypeptide,component (e.g., RNAi, siRNAs, antisense, a nucleic acids encoding a SIRpolypeptide), and other compounds, e.g., as described herein.

In one embodiment, the modulator can be provided to a subject who has oris predisposed to having an age-associated disorder. An “age-associateddisorder” or “age-related disorder” is a disease or disorder whoseincidence is at least 1.5 fold higher among human individuals greaterthan 60 years of age relative to human individuals between the ages of30-40, at the time of filing of this application and in a selectedpopulation of greater than 100,000 individuals. A preferred populationis a United States population. A population can be restricted by genderand/or ethnicity.

In one embodiment, the method includes, e.g., before, during or afterthe providing, evaluating cells of the subject for cytochrome c, e.g.,acetylation state of cytochrome c. The results can be compared toreference results, e.g., from a normal subject or from the subject at adifferent time. The cells that are evaluated can be in a sample from thesubject. For example, the cells can be blood cells, muscle cells, orfibroblasts (e.g., using a cheek swab, etc.).

In one embodiment, the modulator can be provided to a subject who has oris predisposed to having a geriatric disorder. A “geriatric disorder” isa disease or disorder whose incidence, at the time of filing of thisapplication and in a selected population of greater than 100,000individuals, is at least 70% among human individuals that are greaterthan 70 years of age. In one embodiment, the geriatric disorder is adisorder other than cancer or a cardio-pulnonary disorder. A preferredpopulation is a United States population. A population can be restrictedby gender and/or ethnicity.

In one embodiment, the modulator can be provided to a subject who has oris predisposed to having a disorder having an age-associatedsusceptibility factor. A disorder having an “age-associatedsusceptibility factor” refers to a disease or disorder whose causationis mediated by an externality, but whose severity or symptoms aresubstantially increased in human individuals over the age of 60 relativeto human individuals between the ages of 30-40, at the time of filing ofthis application and in the United States population. For example,pneumonia is caused by pathogens, but the severity of the disease isgreater in humans over the age of 60 relative to human individualsbetween the ages of 30-40.

In one embodiment, the modulator can be provided to a subject who has oris predisposed to having a neoplastic disorder or an age-associatedneoplastic disorder. A “neoplastic disorder” is a disease or disordercharacterized by cells that have the capacity for autonomous growth orreplication, e.g., an abnormal state or condition characterized byproliferative cell growth. An “age-associated neoplastic disorder” is aneoplastic disorder that is also an age-associated disorder.

In one embodiment, the modulator can be provided to a subject who has oris predisposed to having a non-neoplastic disorder or an age-associatednon-neoplastic disorder. A “non-neoplastic disorder” is a disease ordisorder that is not characterized by cells that have the capacity forautonomous growth or replication. An “age-associated non-neoplasticdisorder” is a non-neoplastic disorder that is also an age-associateddisorder.

In one embodiment, the modulator can be provided to a subject who has oris predisposed to having a neurological disorder or an age-associatedneurological disorder. A “neurological disorder” is a disease ordisorder characterized by an abnormality or malfunction of neuronalcells or neuronal support cells (e.g., glia or muscle). The disease ordisorder can affect the central and/or peripheral nervous system.Exemplary neurological disorders include neuropathies, skeletal muscleatrophy, and neurodegenerative diseases, e.g., a neurodegenerativedisease caused at least in part by polyglutamine aggregation. Exemplaryneurodegenerative diseases include: Alzheimer's, Amyotrophic LateralSclerosis (ALS), and Parkinson's disease. An “age-associatedneurological disorder is a neurological disorder that is also anage-associated disorder.

In one embodiment, the modulator can be provided to a subject who has oris predisposed to having a cardiovascular disorder or an age-associatedcardiovascular disorder. A “cardiovascular disorder” is a disease ordisorder characterized by an abnormality or malfunction of thecardiovascular system, e.g., heart, lung, or blood vessels. Exemplarycardiovascular disorders include: cardiac dysrhythmias, chroniccongestive heart failure, ischemic stroke, coronary artery disease andcardiomyopathy. An “age-associated cardiovascular disorder is acardiovascular disorder that is also an age-associated disorder.

In one embodiment, the modulator can be provided to a subject who has oris predisposed to having a metabolic disorder or an age-associatedmetabolic disorder. A “metabolic disorder” is a disease or disordercharacterized by an abnormality or malfunction of metabolism. Onecategory of metabolic disorders are disorders of glucose or insulinmetabolism An “age-associated metabolic disorder is a metabolic disorderthat is also an age-associated disorder.

In one embodiment, the modulator can be provided to a subject who has oris predisposed to having a dermatological disorder, a dermatologicaltissue condition, or an age-associated dermatological disorder or tissuecondition. A “dermatological disorder” is a disease or disordercharacterized by an abnormality or malfunction of the skin. A“dermatological tissue condition” refers to the skin and any underlyingtissue (e.g., support tissue) which contributes to the skins functionand/or appearance, e.g., cosmetic appearance.

Exemplary diseases and disorders that are relevant to certainimplementations include: cancer (e.g., breast cancer, colorectal cancer,CCL, CML, prostate cancer); skeletal muscle atrophy; adult-onsetdiabetes; diabetic nephropathy, neuropathy (e.g., sensory neuropathy,autonomic neuropathy, motor neuropathy, retinopathy); obesity; boneresorption; age-related macular degeneration, ALS, Alzheimer's, Bell'sPalsy, atherosclerosis, cardiovascular disorders (e.g., cardiacdysrhythmias, chronic congestive heart failure, ischemic stroke,coronary artery disease and cardiomyopathy), chronic renal failure, type2 diabetes, ulceration, cataract, presbiopia, glomerulonephritis,Guillan-Barre syndrome, hemorrhagic stroke, short-term and long-termmemory loss, rheumatoid arthritis, inflammatory bowel disease, multiplesclerosis, SLE, Crohn's disease, osteoarthritis, Parkinson's disease,pneumonia, and urinary incontinence. In addition, many neurodegenerativedisorders and disorders associated with protein aggregation (e.g., otherthan polyglutamine aggregation) or protein misfolding can also beage-related. Symptoms and diagnosis of diseases are well known tomedical practitioners. The compositions may also be administered toindividuals being treated by other means for such diseases, for example,individuals being treated with a chemotherapeutic (e.g., and havingneutropenia, atrophy, cachexia, nephropathy, neuropathy) or an electivesurgery.

Definitions

A Sir protein, derivative, and functional domains thereof arecollectively referred to as “SIR polypeptides” or “SIR proteins”. “SIRproteins” and “SIR polypeptides” are used interchangeably herein andrefer to members of the Silent Information Regulator (SIR) 2 family ofgenes. The SIR family includes SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6,and SIRT7. The term “SIRT1 proteins” or “SIRT1 polypeptides” refers to apolypeptide that is at least 25% identical to the 250 amino acidconserved SIRT1 catalytic domain, amino acid residues 258 to 451 of SEQID NO:2. SEQ ID NO: 1 depicts the amino acid sequence of human SIRT1. Inpreferred embodiments, a SIRT1 polypeptide can be at least 30, 40, 50,60, 70, 80, 85, 90, 95, 99% homologous to SEQ ID NO:1. In otherembodiments, the SIRT1 polypeptide can be a fragment, e.g., a fragmentof SIRT1 capable of one or more of: deacetylating a substrate in thepresence of NAD and/or a NAD analog and capable of binding a targetprotein, e.g., a cytochrome c. Such functions can be evaluated, e.g., bythe methods described herein. The term “SIRT2 proteins” or “SIRT2polypeptides” refers to a polypeptide at least 30, 40, 50, 60, 70, 80,85, 90, 95, 99% homologous to SEQ ID NO:2. SEQ ID NO:2 depicts the aminoacid sequence of human SIRT2. In other embodiments, the SIRT2polypeptide can be a fragment, e.g., a fragment of SIRT2 capable of oneor more of: deacetylating a substrate in the presence of NAD and/or aNAD analog and capable of binding a target protein, e.g., a cytochromec. Such functions can be evaluated, e.g., by the methods describedherein. The term “SIRT3 proteins” or “SIRT3 polypeptides” refers to apolypeptide at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 99% homologousto SEQ ID NO:3. SEQ ID NO:3 depicts the amino acid sequence of humanSIRT3. In other embodiments, the SIRT3 polypeptide can be a fragment,e.g., a fragment of SIRT3 capable of one or more of: deacetylating asubstrate in the presence of NAD and/or a NAD analog and capable ofbinding a target protein, e.g., a cytochrome c. Such functions can beevaluated, e.g., by the methods described herein. The term “SIRT4proteins” or “SIRT4 polypeptides” refers to a polypeptide at least 30,40, 50, 60, 70, 80, 85, 90, 95, 99% homologous to SEQ ID NO:4. SEQ IDNO:4 depicts the amino acid sequence of human SIRT4. In otherembodiments, the SIRT4 polypeptide can be a fragment, e.g., a fragmentof SIRT4 capable of one or more of: deacetylating a substrate in thepresence of NAD and/or a NAD analog and capable of binding a targetprotein, e.g., a cytochrome c. Such functions can be evaluated, e.g., bythe methods described herein. The term “SIRT5 proteins” or “SIRT5polypeptides” refers to a polypeptide at least 30, 40, 50, 60, 70, 80,85, 90, 95, 99% homologous to SEQ ID NO:5. SEQ ID NO:5 depicts the aminoacid sequence of human SIRT5. In other embodiments, the SIRT5polypeptide can be a fragment, e.g., a fragment of SIRT5 capable of oneor more of: deacetylating a substrate in the presence of NAD and/or aNAD analog and capable of binding a target protein, e.g., a cytochromec. Such functions can be evaluated, e.g., by the methods describedherein. The term “SIRT6 proteins” or “SIRT6 polypeptides” refers to apolypeptide at least 30, 40, 50, 60, 70, 80, 85, 90, 95, 99% homologousto SEQ ID NO:6. SEQ ID NO:6 depicts the amino acid sequence of humanSIRT6. In other embodiments, the SIRT6 polypeptide can be a fragment,e.g., a fragment of SIRT6 capable of one or more of: deacetylating asubstrate in the presence of NAD and/or a NAD analog and capable ofbinding a target protein, e.g., a cytochrome c. Such functions can beevaluated, e.g., by the methods described herein. The term “SIRT7proteins” or “SIRT7 polypeptides” refers to a polypeptide at least 30,40, 50, 60, 70, 80, 85, 90, 95, 99% homologous to SEQ ID NO:7. SEQ IDNO:7 depicts the amino acid sequence of human SIRT7. In otherembodiments, the SIRT7 polypeptide can be a fragment, e.g., a fragmentof SIRT7 capable of one or more of: deacetylating a substrate in thepresence of NAD and/or a NAD analog and capable of binding a targetprotein, e.g., a cytochrome c. Such functions can be evaluated, e.g., bythe methods described herein. The sequences of human SIRT1, SIRT2,SIRT3, SIRT4 and SIRT5 are also disclosed, e.g., in Frye et al. (1999)Biochem. Biophys. Res. Comm. 260(1):273-279, and the sequences of humanSIRT6 and SIRT7 are disclosed, e.g., in Frye et al. (2000) Biochem.Biophys. Res. Comm. 273(2):793-798, the contents of which areincorporated herein by reference. In some embodiments, the SIRpolypeptide can be a “full length” SIR polypeptide. The term “fulllength” as used herein refers to a polypeptide that has at least thelength of a naturally occurring SIR polypeptide (or other proteindescribed herein). A “full length” SIR polypeptide or a fragment thereofcan also include other sequences, e.g., a purification tag., or otherattached compounds, e.g., an attached fluorophore, or cofactor. The term“SIR polypeptides” can also include sequences or variants that includeone or more substitutions, e.g., between one and ten substitutions, withrespect to a naturally occurring Sir2 family member. In preferredembodiments, a human SIR polypeptide can vary from SEQ ID NO:1, 2, 3, 4,5, 6, or 7 by at least 1, 2, 3, 4, 5, 10, 15, but preferably not morethan 20 to 50 amino acid residues, e.g., it can vary by at least 1, 2,3, 4, 5, 10, 15 substitutions, e.g., conservative substitutions. In oneembodiment, the variation is a naturally occurring variation. In otherembodiments, the SIR polypeptide is encoded by a nucleic acid whichhybridizes under stringent conditions to a nucleic acid encoding theamino acid sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, or 7. Thisapplication also incorporates by reference U.S. Ser. No. 10/191,121, forall purposes. This application also includes a listing of exemplary SIRsequences.

The following are exemplary SIR sequences: >sp|Q96EB6|SIR1_HUMANNAD-dependent deacetylase sirtuin 1 (EC 3.5.1.-) (hSIRT1) (hSIR2)(SIR2-like protein 1) - Homo sapiens (Human). (SEQ ID NO :1)MADEAALALQPGGSPSAAGADREAASSPAGEPLRKRPRRDGPGLERSPGEPGGAAPEREVPAAARGCPGAAAAALWREAEAEAAAAGGEQEAQATAAAGEGDNGPGLQGPSREPPLADNLYDEDDDDEGEEEEEAAAAAIGYRDNLLFGDEIITNGFHSCESDEEDRASHASSSDWTPRPRIGPYTFVQQHLMIGTDPRTILKDLLPETIPPPELDDMTLWQIVINILSEPPKRKKRKDINTIEDAVKLLQECKKIIVLTGAGVSVSCGIPDFRSRDGIYARLAVDFPDLPDPQAMFDIEYFRKDPRPFFKFAKEIYPGQFQPSLCHKFIALSDKEGKLLRNYTQNIDTLEQVAGIQRIIQCHGSFATASCLICKYKVDCEAVRGDIFNQVVPRCPRCPADEPLAIMKPEIVFFGENLPEQFHRAMKYDKDEVDLLIVIGSSLKVRPVALIPSSIPHEVPQILINREPLPHLHFDVELLGDCDVIINELCHRLGGEYAKLCCNPVKLSEITEKPPRTQKELAYLSELPPTPLHVSEDSSSPERTSPPDSSVIVTLLDQAAKSNDDLDVSESKGCMEEKPQEVQTSRNVESIAEQMENPDLKNVGSSTGEKNERTSVAGTVRKCWPNRVAKEQISRRLDGNQYLFLPPNRYIFHGAEVYSDSEDDVLSSSSCGSNSDSGTCQSPSLEEPMEDESEIEEFYNGLEDEPDVPERAGGAGFGTDGDDQEAINEAISVKQEVTDMNYPSNKS >sp|Q8IXJ6|SIR2_HUMAN NAD-dependentdeacetylase sirtuin 2 (EC 3.5.1.-) (SIR2-like) (SIR2- like protein 2) -Homo sapiens (Human). (SEQ ID NO:2)MAEPDPSHPLETQAGKVQEAQDSDSDSEGGAAGGEADMDFLRNLFSQTLSLGSQKERLLDELTLEGVARYMQSERCRRVICLVGAGISTSAGIPDFRSPSTGLYDNLEKYHLPYPEAIFEISYFKKHPEPFFALAKELYPGQFKPTICHYFMRLLKDKGLLLRCYTQNIDTLERIAGLEQEDLVEAHGTFYTSHCVSASCRHEYPLSWMKEKIFSEVTPKCEDCQSLVKPDIVFFGESLPARFFSCMQSDFLKVDLLLVMGTSLQVQPFASLISKAPLSTPRLLINKEKAGQSDPFLGMIMGLGGGMDFDSKKAYRDVAWLGECDQGCLALAELLGWKKELEDLVRREHASIDAQSGAGVPNPSTSASPKKSPPPAKDEARTTEREKPQ >sp|Q9NTG7|SIR3_HUMAN NAD-dependentdeacetylase sirtuin 3, mitochondrial precursor (EC 3.5.1.-) (SIR2-likeprotein 3) (hSIRT3) - Homo sapiens (Human). (SEQ ID NO: 3)MAFWGWRAAAALRLWGRVVERVEAGGGVGPFQACGCRLVLGGRDDVSAGLRGSHGARGEPLDPARPLQRPPRPEVPRAFRRQPRAAAPSFFFSSIKGGRRSISFSVGASSVVGSGGSSDKGKLSLQDVAELIRARACQRVVVMVGAGISTPSGIPDFRSPGSGLYSNLQQYDLPYPEAIFELPFFFHNPKPFFTLAKELYPGNYKPNVTHYFLRLLHDKGLLLRLYTQNIDGLERVSGIPASKLVEAHGTFASATCTVCQRPFPGEDIRADVMADRVPRCPVCTGVVKPDIVFFGEPLPQRFLLHVVDFPMADLLLILGTSLEVEPFASLTEAVRSSVPRLLINRDLVGPLAWHPRSRDVAQLGDVVHGVESLVELLGWTEEMRDLVQRETGKLDGPDK >sP|Q9Y6E7|SIR4_HUMANNAD-dependent deacetylase sirtuin 4 (EC 3.5.1.-) (SIR2-like protein 4) -Homo sapiens (Human). (SEQ ID NO:4)MKMSFALTFRSAKGRWIANPSQPCSKASIGLFVPASPPLDPEKVKELQRFITLSKRLLVMTGAGISTESGIPDYRSEKVGLYARTDRRPIQHGDFVRSAPIRQRYWARNFVGWPQFSSHQPNPAHWALSTWEKLGKLYWLVTQNVDALHTKAGSRRLTELHGCMDRVLCLDCGEQTPRGVLQERFQVLNPTWSAEAHGLAPDGDVFLSEEQVRSFQVPTCVQCGGHLKPDVVFFGDTVNPDKVDFVHKRVKEADSLLVVGSSLQVYSGYRFILTAWEKKLPIAILNIGPTRSDDLACLKLNSRCGELLPLIDPC >sp|Q9NXA8|SIR5_HUMAN NAD-dependent deacetylase sirtuin 5(EC 3.5.1.-) (SIR2-like protein 5) - Homo sapiens (Human). (SEQ ID NO:5)MRPLQIVPSRLISQLYCGLKPPASTRNQICLKMARPSSSMADFRKFFAKAKHIVIISGAGVSAESGVPTFRGAGGYWRKWQAQDLATPLAFAHNPSRVWEFYHYRREVMGSKEPNAGHRAIAECETRLGKQGRRVVVITQNIDELHRKAGTKNLLEIHGSLFKTRCTSCGVVAENYKSPICPALSGKGAPEPGTQDASIPVEKLPRCEEAGCGGLLRPHVVWFGENLDPAILEEVDRELAHCDLCLVVGTSSVVYPAAMFAPQVAARGVPVAEFNTETTPATNRFRFHFQGPCGTTLPEALACHENETVS >sp |Q8N6T7|SIR6_HUMAN NAD-dependent deacetylase sirtuin 6(EC 3.5.1.-) (SIR2-like protein 6) - Homo sapiens (Human). (SEQ ID NO:6)MSVNYAAGLSPYADKGKCGLPEIFDPPEELERKVWELARLVWQSSSVVFHTGAGISTASGIPDFRGPHGVWTMEERGLAPKFDTTFESARPTQTHMALVQLERVGLLRFLVSQNVDGLHVRSGFPRDKLAELHGNMFVEECAKCKTQYVRDTVVGTMGLKATGRLCTVAKARGLRACRGELRDTILDWEDSLPDRDLALADEASRNADLSITLGTSLQIRPSGNLPLATKRRGGRLVIVNLQPTKHDRHADLRIHGYVDEVMTRLMKHLGLEIPAWDGPRVLERALPPLPRPPTPKLEPKEESPTRINGSIPAGPKQEPCAQHNGSEPASPKRERPTSPAPHRPPKRVKAKAVPS >sp|Q9NRC8|SIR7_HUMANNAD-dependent deacetylase sirtuin 7 (EC 3.5.1.-) (SIR2-like protein 7) -Homo sapiens (Human). (SEQ ID NO :7)MAAGGLSRSERKAAERVRRLREEQQRERLRQVSRILRKAAAERSAEEGRLLAESADLVTELQGRSRRREGLKRRQEEVCDDPEELRGKVRELASAVRNAKYLVVYTGAGISTAASIPDYRGPNGVWTLLQKGRSVSAADLSEAEPTLTHMSITRLHEQKLVQHVVSQNCDGLHLRSGLPRTAISELHGNMYIEVCTSCVPNREYVRVFDVTERTALHRHQTGRTCHKCGTQLRDTIVHFGERGTLGQPLNWEAATEAASRADTILCLGSSLKVLKKYPRLWCMTKPPSRRPKLYIVNLQWTPKDDWAALKLHGKCDDVMRLLMAELGLEIPAYSRWQDPIFSLATPLRAGEEGSHSRKSLCRSREEAPPGDRGAPLSSAPILGGWFGRGCTKRTKRKKVT

The term “SIR polypeptide” also includes homologs of the various humanSIR polypeptides from other species, e.g., other mammalian species,including the murine homologs. A “SIR activity” refers to one or moreactivity of a SIR polypeptide, e.g., deacetylation of a substrate, e.g.,cytochrome c, (e.g., in the presence of a cofactor such as NAD and/or anNAD analog) and binding of a target protein, e.g., cytochrome c or anuclear protein, e.g., a transcription factor.

As used herein, the term “nucleic acid molecule” includes DNA molecules(e.g., a cDNA or genomic DNA), RNA molecules (e.g., an mRNA) and analogsof the DNA or RNA. A DNA or RNA analog can be synthesized fromnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded, e.g., double-stranded DNA or a double-stranded RNA.Nucleic acid molecules encoding the SIR polypeptides or proteins arecollectively referred to as “SIR nucleic acids”. Such nucleic acidsinclude naturally occurring genomic and cDNA sequences, naturallyoccurring variants, and synthetic sequences (e.g., codon-optimizedcoding sequences). The polypeptide may include one or more unnaturalamino acids. Typically, the polypeptide includes only natural aminoacids. The term “peptide” refers to a polypeptide that is between threeand thirty-two amino acids in length. A protein or polypeptide can alsoinclude one or more modifications, e.g., a glycosylation, amidation,phosphorylation, and so forth.

The term “isolated nucleic acid molecule” or “purified nucleic acidmolecule” includes nucleic acid molecules that are separated from othernucleic acid molecules present in the natural source of the nucleicacid. For example, with regards to genomic DNA, the term “isolated”includes nucleic acid molecules which are separated from the chromosomewith which the genomic DNA is naturally associated. In some embodiments,an “isolated” nucleic acid is free of sequences which naturally flankthe nucleic acid (i.e., sequences located at the 5′ and/or 3′ ends ofthe nucleic acid) in the genomic DNA of the organism from which thenucleic acid is derived. For example, in various embodiments, theisolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5′ and/or 3′ nucleotide sequenceswhich naturally flank the nucleic acid molecule in genomic DNA of thecell from which the nucleic acid is derived. Examples of flankingsequences include adjacent genes, transposons, and regulatory sequences.Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule,can be substantially free of other cellular material, of culture mediumwhen produced by recombinant techniques, or of chemical precursors orother chemicals when chemically synthesized.

As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which isincorporated by reference. Aqueous and nonaqueous methods are describedin that reference and either can be used. Specific hybridizationconditions referred to herein are as follows: 1) low stringencyhybridization conditions in 6× sodium chloride/sodium citrate (SSC) atabout 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at50° C. (the temperature of the washes can be increased to 55° C. for lowstringency conditions); 2) medium stringency hybridization conditions in6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1%SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC atabout 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65°C.; and preferably 4) very high stringency hybridization conditions are0.5 M sodium phosphate, 7% SDS at 65° C., followed by one or more washesat 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are thepreferred conditions and the ones that should be used unless otherwisespecified. Methods of the invention can include use of an isolatednucleic acid molecule of the invention that hybridizes under astringency condition described herein to a sequence described herein oruse of a polypeptide encoded by such a sequence, e.g., the molecule canbe a naturally occurring variant.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs inNature. For example a naturally occurring nucleic acid molecule canencode a natural protein.

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules which include at least an open reading frame encoding aprotein or subunit, derivative, or functional domain thereof. The genecan optionally further include non-coding sequences, e.g., regulatorysequences and introns.

An “isolated” or “purified” polypeptide or protein is substantially freeof cellular material or other contaminating proteins from the cell ortissue source from which the protein is derived, or substantially freefrom chemical precursors or other chemicals when chemically synthesized.“Substantially free” means that the protein of interest in thepreparation is at least 10% pure. In an embodiment, the preparation ofthe protein has less than about 30%, 20%, 10% and more preferably 5% (bydry weight), of a contaminating component (e.g., a protein not ofinterest, chemical precursors, and so forth). When the protein orbiologically active portion thereof is recombinantly produced, it isalso preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, more preferably less than about10%, and most preferably less than about 5% of the volume of the proteinpreparation. The invention includes isolated or purified preparations ofat least 0.01, 0.1, 1.0, and 10 milligrams in dry weight.

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of protein without abolishing orsubstantially altering activity, e.g., the activity is at least 20%,40%, 60%, 70% or 80% of wild-type. An “essential” amino acid residue isa residue that, when altered from the wild-type sequence results inabolishing activity such that less than 20% of the wild-type activity ispresent. Conserved amino acid residues are frequently predicted to beparticularly unamenable to alteration.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a protein is preferablyreplaced with another amino acid residue from the same side chainfamily. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity. Followingmutagenesis, the encoded protein can be expressed recombinantly and theactivity of the protein can be determined.

As used herein, a “biologically active portion” or a “functional domain”of a protein includes a fragment of a protein of interest whichparticipates in an interaction, e.g., an intramolecular or aninter-molecular interaction, e.g., a binding or catalytic interaction.An inter-molecular interaction can be a specific binding interaction oran enzymatic interaction (e.g., the interaction can be transient and acovalent bond is formed or broken). An inter-molecular interaction canbe between the protein and another protein, between the protein andanother compound, or between a first molecule and a second molecule ofthe protein (e.g., a dimerization interaction). Biologically activeportions/functional domains of a protein include peptides comprisingamino acid sequences sufficiently homologous to or derived from theamino acid sequence of the protein which include fewer amino acids thanthe full length, natural protein, and exhibit at least one activity ofthe natural protein. Biological active portions/functional domains canbe identified by a variety of techniques including truncation analysis,site-directed mutagenesis, and proteolysis. Mutants or proteolyticfragments can be assayed for activity by an appropriate biochemical orbiological (e.g., genetic) assay. In some embodiments, a functionaldomain is independently folded. Typically, biologically active portionscomprise a domain or motif with at least one activity of the protein,e.g., a SIR core catalytic domain. A biologically activeportion/functional domain of a protein can be a polypeptide which is,for example, 10, 25, 50, 100, 200 or more amino acids in length.Biologically active portions/functional domain of a protein can be usedas targets for developing agents which modulate apoptosis.

A variety of methods can be used to identify a SIR family member. Forexample, a known amino acid sequence of a human SIR polypeptide, e.g.,any of human SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7 can besearched against the GenBank™ sequence databases (National Center forBiotechnology Information, National Institutes of Health, Bethesda Md.),e.g., using BLAST; against Pfam database of HMMs (Hidden Markov Models)(using default parameters for Pfam searching; against the SMARTdatabase; or against the ProDom database. For example, the hmmsfprogram, which is available as part of the HMMER package of searchprograms, is a family specific default program for MILPAT0063 and ascore of 15 is the default threshold score for determining a hit.Alternatively, the threshold score for determining a hit can be lowered(e.g., to 8 bits). A description of the Pfam database can be found inSonhammer et al. (1997) Proteins 28(3):405-420 and a detaileddescription of HMMs can be found, for example, in Gribskov et al. (1990)Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad.Sci. USA 84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531;and Stultz et al. (1993) Protein Sci. 2:305-314. The SMART database(Simple Modular Architecture Research Tool, EMBL, Heidelberg, Del.) ofHMMs as described in Schultz et al. (1998), Proc. Natl. Acad. Sci. USA95:5857 and Schultz et al. (200) Nucl. Acids Res 28:231. The SMARTdatabase contains domains identified by profiling with the hidden Markovmodels of the HMMer2 search program (R. Durbin et al. (1998) Biologicalsequence analysis: probabilistic models of proteins and nucleic acids.Cambridge University Press). The database also is annotated andmonitored. The ProDom protein domain database consists of an automaticcompilation of homologous domains. (Corpet et al. (1999), Nucl. AcidsRes. 27:263-267) Current versions of ProDom are built using recursivePSI-BLAST searches (Altschul et al. (1997) Nucleic Acids Res.25:3389-3402; Gouzy et al. (1999) Computers and Chemistry 23:333-340.)of the SWISS-PROT 38 and TREMBL protein databases. The databaseautomatically generates a consensus sequence for each domain.

Calculations of homology or sequence identity between sequences (theterms are used interchangeably herein) are performed as follows.

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, 60%, and even more preferably at least 70%,80%, 90%, 100% of the length of the reference sequence. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”).

The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporatedinto the GAP program in the GCG software package, using either a Blossum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6,or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet anotherpreferred embodiment, the percent identity between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage, using the NWSgapdna. CMP matrix and a gap weight of 40, 50, 60,70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularlypreferred set of parameters (and the one that should be used unlessotherwise specified) are a Blossum 62 scoring matrix with a gap penaltyof 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of Meyers and Miller ((1989) CABIOS,4:11-17) which has been incorporated into the ALIGN program (version2.0), using a PAM120 weight residue table, a gap length penalty of 12and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to nucleic acidmolecules of the invention. BLAST protein searches can be performed withthe XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to protein molecules of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402.When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) can be used.

Some polypeptides of the present invention can have an amino acidsequence substantially identical to an amino acid sequence describedherein. In the context of an amino acid sequence, the term“substantially identical” is used herein to refer to a first amino acidthat contains a sufficient or minimum number of amino acid residues thatare i) identical to, or ii) conservative substitutions of aligned aminoacid residues in a second amino acid sequence such that the first andsecond amino acid sequences can have a common structural domain and/orcommon functional activity. Methods of the invention can include use ofa polypeptide that includes an amino acid sequence that contains astructural domain having at least about 60%, or 65% identity, likely 75%identity, more likely 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99%identity to a domain of a polypeptide described herein.

In the context of nucleotide sequence, the term “substantiallyidentical” is used herein to refer to a first nucleic acid sequence thatcontains a sufficient or minimum number of nucleotides that areidentical to aligned nucleotides in a second nucleic acid sequence suchthat the first and second nucleotide sequences encode a polypeptidehaving common functional activity, or encode a common structuralpolypeptide domain or a common functional polypeptide activity. Methodsof the invention can include use of a nucleic acid that includes aregion at least about 60%, or 65% identity, likely 75% identity, morelikely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identityto a nucleic acid sequence described herein, or use of a protein encodedby such nucleic acid.

A “purified preparation of cells”, as used herein, refers to an in vitropreparation of cells. In the case cells from multicellular organisms(e.g., plants and animals), a purified preparation of cells is a subsetof cells obtained from the organism, not the entire intact organism. Inthe case of unicellular microorganisms (e.g., cultured cells andmicrobial cells), it consists of a preparation of at least 10% and morepreferably 50% of the subject cells.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

A “small organic molecule” is an organic molecule of having a molecularweight of less than 5, 2, 1, or 0.5 kDa. In many embodiments, such smallmolecules do not include a peptide bond or a phosphodiester bond. Forexample, they can be non-polymeric. In some embodiments, the moleculehas a molecular weight of at least 50, 100, 200, or 400 Daltons.

“Binding affinity” refers to the apparent dissociation constant orK_(D). A ligand may, for example, have a binding affinity of at least10⁻⁵, 10⁻⁶, 10⁻⁷ or 10⁻⁸ M for a particular target molecule. Higheraffinity binding of a ligand to a first target relative to a secondtarget can be indicated by a smaller numerical value K_(D) ¹ for bindingthe first target than the numerical value K_(D) ² for binding the secondtarget. In such cases the ligand has specificity for the first targetrelative to the second target. The agent may bind specifically to thetarget, e.g., with an affinity that is at least 2, 5, 10, 100, or 1000better than for a non-target. For example, an agent can bind to a SIRpolypeptide with a K_(d) of less than 10⁻⁵, 10⁻⁶, 10⁻⁷ or 10⁻⁸ M. If theagent binds specifically, it may be binding to a protein, e.g., to a SIRpolypeptide with a K_(d) of greater than 2, 5, 10, 100, or 1000 times10⁻⁵, 10⁻⁶, 10⁻⁷ or 10⁻⁸ M, as appropriate.

Binding affinity can be determined by a variety of methods includingequilibrium dialysis, equilibrium binding, gel filtration, ELISA, orspectroscopy (e.g., using a fluorescence assay). These techniques can beused to measure the concentration of bound and free ligand as a functionof ligand (or target) concentration. The concentration of bound ligand([Bound]) is related to the concentration of free ligand ([Free]) andthe concentration of binding sites for the ligand on the target where(N) is the number of binding sites per target molecule by the followingequation:[Bound]=N·[Free]/((1/Ka)+[Free])

A “cytochrome c activity” refers to one or more activities of cytochromec, e.g., cytochrome c mediated apoptosis and/or mitochondrialrespiration.

A “cytochrome c polypeptide” refers to a full length cytochrome c or afragment thereof, e.g., having at least 3 amino acids, e.g., between3-50, 3-20, 3-15, 3-10, 5-20, 5-12 5-10, or 6-12. An exemplarycytochrome c polypeptide is a human cytochrome c polypeptide which caninclude the following sequence or a fragment thereof: GDVEKGKKIFIMKCSQCHTV EKGGKHKTGP NLHGLFGRKT GQAPGYSYTA ANKNKGIIWG EDTLMEYLENPKKYIPGTKM IFVGIKKKEE RADLIAYLKK ATNE

Exemplary fragments include a lysine. A cytochrome c polypeptide (e.g.,full length or a fragment) can be acetylated, e.g., at one or more ofthe lysines.

One exemplary cytochrome c fragment is able to bind heme. Thefragment-heme complex can have spectroscopic properties similar to afull length cytochrome c bound to heme. The fragment-heme complex can beacetylated.

“Modulating cytochrome c activity” refers to increasing or decreasingcytochrome c activity, e.g., cytochrome c-mediated apoptosis, cell cyclearrest, and/or senescence, e.g. by altering the acetylation status ofcytochrome c.

The term “chronological age” as used herein refers to time elapsed sincea preselected event, such as biological age” as conception, a definedembryological or fetal stage, or, more preferably, birth.

In contrast, the term “biological age” refers to manifestations of thepassage of time that is not linearly fixed with the amount of timeelapsed. The manifestations of biological aging are varied and maydepend on the species of organism, environmental conditions, and, asdiscussed herein, genotype. Exemplary manifestations of biological agingin mammals include endocrine changes (for example, puberty, menses,changes in fertility or fecundity, menopause, and secondary sexcharacteristics, such as balding,), metabolic changes (for example,changes in appetite and activity), and immunological changes (forexample, changes in resistance to disease). The appearance of mammalsalso changes with biological age, for example, graying of hair,wrinkling of skin, and so forth. With respect to a different class ofanimals, the nematode C. elegans also has manifestations of biologicalaging, for example, changes in fecundity, activity, responsiveness tostimuli, and appearance (e.g., change in intestinal autofluorescence andflaccidity). In many cases, the remaining potential lifespan of anindividual is a function of its biological age.

The term “average lifespan” refers to the average of the age of death ofa cohort of organisms. In some cases, the “average lifespan” is assessedusing a cohort of genetically identical organisms under controlledenvironmental conditions. Deaths due to mishap are discarded. Forexample, with respect to a nematode population, hermaphrodites that dieas a result of the “bag of worms” phenotype are typically discarded. Avariety of criteria can be used to determine whether organisms are ofthe “same” chronological age for the comparative analysis. Typically,the degree of accuracy required is a function of the average lifespan ofa wild type organism. For example, for the nematode C. elegans, forwhich the laboratory wild type strain N2 lives an average of about 16days under some controlled conditions, organisms of the same age mayhave lived for the same number of days. For mice, organism of the sameage may have lived for the same number of weeks or months; for primatesor humans, the same number of years (or within 2, 3, or 5 years); forDrosophila, the same number of weeks; and so forth.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims. All patents, patentapplications, inclusive of Ser. No. 60/433,096, and references citedherein are incorporated in their entirety by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing recombinant GST-SIRT2 deacetylation ofacetylated cytochrome c in the presence or absence of NAD.

FIG. 2 is a graph showing recombinant GST-SIRT3 deacetylation ofacetylated cytochrome c in the presence or absence of NAD.

FIG. 3 is a graph showing deacetylation of chemically acetylatedcytochrome c by SIRT3 enzyme derived from pull downs of 293T cellextracts in nicotinamide release assays of SIRT3-GFP or GFP (control)proteins. The substrate is indicated in parentheses (cytochrome c orhistone H4).

FIG. 4 is a graph depicting deacetylation of chemically acetylatedcytochrome c by SIRT1-7 enzymes derived from pull downs of 293T cellextracts in nicotinamide release assays. The substrate cytochrome c isindicated in parentheses.

DETAILED DESCRIPTION OF THE INVENTION

Control of cytochrome c acetylation, e.g., with a SIR polypeptide,enables interventions that modulate lifespan regulation and cellproliferation, e.g., by modulating apoptosis and/or mitochondrialfunction such as respiration. Agents which alter lifespan regulation ofa cell or organism are identified by screening for compounds thatregulate cytochrome c acetylation status, to, e.g., modulate cytochromec-mediated apoptosis. The agents so-identified and compounds known toalter interaction of cytochrome c and proteins having acetylation ordeacetylation activity and/or otherwise alter acetylation status can beadministered to a subject, e.g., to alter an apoptotic program orlifespan regulation in the subject or to affect a longevity-associateddisorder, or a risk, symptom, predisposition thereof. In other aspects,agents which alter cell proliferation (e.g., decrease cellproliferation) are identified by screening compounds that reduceacetylation of cytochrome c and/or increase cytochrome c-mediatedapoptosis.

For example, lifespan regulation can be modulated to enhance, increase,or otherwise favor increased lifespan. A method to increase lifespan caninclude modulating components of the cytochrome c-mediated apoptosispathway to: increase acetylation of cytochrome c; decrease interaction,e.g., binding, of a polypeptide having deacetylation activity, e.g., aSIR polypeptide, to cytochrome c; decrease interaction, e.g., binding,between cytochrome c and Apaf-1; and/or decrease cytochrome c-mediatedapoptosis of a cell, to thereby favor increased lifespan. Relatedmethods can be used to activate physiological processes in an organismthat are associated with an organism of reduced chronological age, e.g.,a genetically identical or genetically normal organism of reducedchronological age.

In other aspects, cell proliferation can be modulated to decrease,inhibit or prevent cell proliferation in disorders characterized byunwanted cell proliferation such as cancers. As described herein,polypeptides having deacetylation activity can interact with humancytochrome c protein to deacetylate cytochrome c. A functionalconsequence of this deacetylation is an increase of the cytochrome c'sinteraction with Apaf-1 and its apoptotic activity. Stress on a cell canresult in release of cytochrome c from the mitochondria into the cytosolwhere in its non-acetylated form, it can interact with Apaf-1.Interaction of cytochrome c with Apaf-1 induces events (e.g., theactivation of caspases) which can eventually result in apoptosis of thecell. While not wishing to be bound by theory, deacetylation ofcytochrome c may enhances cytochrome c's ability to interact with Apaf-1and induce cytochrome c-mediated apoptotic response. The formation oftumors is a multistep process requiring progressive accumulation ofgenetic alterations. Thus, cytochrome c release from the mitochondriadue to stress, e.g., oncogenic stress, can play an important role incancer by inducing apoptosis of damaged cells. A consequence of loss ofpro apoptotic activity is the accumulation of the half-dozen or somutations necessary for a cell to become carcinogenic. A secondconsequence may be uncontrolled cell growth, e.g., metastases, of thecell. In order to enhance or increase cytochrome c mediated apoptosis inthese damaged cells, deacetylation of cytochrome c, is, preferably,reduced, inhibited or prevented.

Examples of methods for modulating expression of polypeptides havingacetylation or deacetylation activity and/or activity and/or cytochromec acetylation and/or cytochrome c-mediated apoptotic activity in cellsand organisms are described below, as are method of identifying agentwhich modulate these activities. Many of the screening assays aredescribed with regards to a SIR polypeptide, however, other polypeptidescan also be used in the screening assays.

Members of the cytochrome c-mediated apoptotic pathway includepolypeptides having deacetylation activity such as Sir polypeptides(e.g., SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and/or SIRT7polypeptides), Apaf-1, caspases, such as caspase 9 and caspase 3. Theinteraction between these members of the cytochrome c-mediated apoptoticpathway is described in detail herein.

Screening Assays

The invention includes methods of screening for a compound, e.g., acompound which interacts with cytochrome c or otherwise effectscytochrome c acetylation status and has an effect on (e.g., induces)apoptosis. Such compounds can be identified as candidates for reducingunwanted cell proliferation in vitro or in vivo (e.g., in a subjecthaving a disorder characterized by unwanted cell proliferation). Themethod can include providing a compound which interacts with cytochromec or a polypeptide having acetylation or deacetylation activity andevaluating the effect of the compound on apoptosis. Compounds whichinteract with cytochrome c or other polypeptides can be identified,e.g., by in vitro or in vivo assays. When both the assay for screening acompound for the ability to interact with cytochrome c or a polypeptideand the assay for determining effect on apoptosis are performed in vivo,e.g., in cell based assays, the assays can be performed in the same ordifferent cells. For example, one or both of the assays can be performedin tissue culture (e.g., BJT cells, 293T cells, MCF-7 cells, H1299cells) or in an organism (e.g., a mammal, e.g., as a human).

In preferred embodiments, the assays are performed in the presence of apolypeptide cofactor, e.g., a SIR polypeptide co-factor, such as NADand/or NAD analogs. In some embodiments, the co-factor is added to thecell culture or in vitro assay, e.g., the NAD and/or an NAD analog canbe placed in sufficient proximity to cause a SIR polypeptide activitysuch as deacetylation. “NAD” refers to nicotinamide adeninedinucleotide. An “NAD analog” as used herein refers to a compound (e.g.,a synthetic or naturally occurring chemical, drug, protein, peptide,small organic molecule) which possesses structural similarity tocomponent groups of NAD (e.g., adenine, ribose and phosphate groups) orfunctional similarity (e.g., deacetylates p53 in the presence of Sir2).For example, an NAD analog can be 3-aminobenzamide or1,3-dihydroisoquinoline (H. Vaziri et al., EMBO J. 16:6018-6033 (1997),the entire teachings of which are hereby incorporated by reference).

Described below are exemplary methods for identifying compounds thatinteract with cytochrome c and/or a polypeptide having acetylation ordeacetylation activity and can have an effect on apoptosis. Preferably,compounds can be identified which interact with, e.g., bind to,cytochrome c or a polypeptide having acetylation or deacetylationactivity, and increase acetylation. Deacetylation of a substrates suchas cytochrome c has been found to increase substrate-induced apoptosis,e.g., cytochrome c induced apoptosis. Cytochrome c can be, for example,the mature protein or a fragment thereof. In a preferred embodiment, thecytochrome c is human cytochrome c. In many instances, such deacetylatedsubstrates may play a role in apoptosis of stressed and/or damagedcells, e.g., DNA damaged cells, e.g., cancer cells. Thus, in someembodiments, it is desirable to identify compounds which interact with apolypeptide having deacetylation activity and increase expression and oractivity of the polypeptide, thereby increasing apoptosis in a cell,e.g., a cancer cell. The phrase “deacetylating a substrate” or“deacetylating cytochrome c” refers to the removal of one or more acetylgroups (e.g., CH₃CO²⁻) from the substrate such as cytochrome c that isacetylated on at least one amino acid residue. Cytochrome c can bedeacetylated in the presence or absence of DNA damage or oxidativecellular stress. The DNA damage can be caused by, for example, ionizingradiation (e.g., 6 Gy of ionizing radiation), or a tumor or some otheruncontrolled cell proliferation. “Acetylation status” refers to thepresence or absence of one or more acetyl groups (e.g., CH₃CO²⁻) at oneor more lysine (K) residues of a substrate, e.g., a transcriptionfactor. For example, the presence of an acetylate groups can be found atone or more of places of the cytochrome sequence depicted in SEQ ID NO.16. “Altering the acetylation state” refers to adding or removing one ormore acetyl groups (e.g., CH₃CO²⁻). For example, adding or removing oneor more acetyl groups of cytochrome c at one or more lysine (K) residuesof SEQ ID NO. 16.

A cytochrome c polypeptide can be monitored using a fluorescence assay.For example, an acetylated cytochrome c polypeptide can be used as asubstrate and monitored, e.g., as described in U.S. 2003-0082668. Forexample, the cytochrome c polypeptide can be prepared as a fluorescentsubstance that includes a fluorescence group and a quencher. Specificexamples include MOAc, Nma (N-methylanthranilic acid), etc. Further, aquencher group should have the characteristic to quench the fluorescenceof a fluorescent group within the same peptide molecule; specificexamples include Dnp, and so on.

In one embodiment, peptides that serve as substrates for deacetylasescan include a peptide with an amino terminal Boc group, amino acidresidues, an acetylated lysine residue, and an MCA group, C terminal tothe lysine (e.g., adjacent). Boc represents a protecting group for theamino group at the N-terminal position of the peptide. MCA is afluorescent substance; and the epsilon-amino group of the flankinglysine residue is acetylated. When the peptides is cleaved at thecarboxyl-terminal side of the lysine residue, they release AMC. Sincethe emission wavelength of the released AMC differs from that ofpeptidyl MCA, the quantity of cleaved substrates can be determined usingthe fluorescence intensity of AMC as an index.

The following assays provide methods (also referred to herein as“evaluating a compound” or “screening a compound”) for identifyingmodulators, i.e., candidate or test compounds (e.g., peptides,peptidomimetics, small molecules or other drugs) which interact withand/or modulate, e.g., have a stimulatory or inhibitory effect on, forexample, cytochrome c acetylation status. Such compounds can be agonistsor antagonists of deacetylation, e.g., agonist or antagonist of a SIRpolypeptide. In preferred embodiments, the screening assays describedherein are used to identify candidates which function as deacetylationantagonists. As described herein, such antagonists can decreaseapoptosis of a cell, which has practical utility, e.g., in altering lifespan regulation. In other preferred embodiment, the screening assaysdescribed herein are used to identify candidates which function asdeacetylation agonists. As described herein, such agonists can increaseapoptosis of a cell, which has practical utility, e.g., in cancer. Someof these assays may be performed in animals, e.g., mammals, in organs,in cells. Others may be performed in animals, e.g., mammals, in organs,in cells, in cell extracts, e.g., purified or unpurified nuclearextracts, intracellular extracts, in purified preparations, in cell-freesystems, in cell fractions enriched for certain components, e.g.,organelles or compounds, or in other systems known in the art. Given theteachings herein and the state of the art, a person of ordinary skill inthe art would be able to choose an appropriate system and assay forpracticing the methods of the present invention.

Some exemplary screening assays for assessing activity or functioninclude one or more of the following features:

-   -   use of a transgenic cell, e.g., with a transgene encoding a        polypeptide having acetylation or deacetylation activity and/or        a cytochrome c polypeptide or mutants thereof;    -   use of a mammalian cell that expresses a polypeptide having        acetylation or deacetylation activity and/or cytochrome c;    -   detection of binding of a labeled compound to a polypeptide        having acetylation or deacetylation activity or cytochrome c        where the compound is, for example, a peptide, protein, antibody        or small organic molecule; e.g., the compound interferes with or        disrupts an interaction between the polypeptide and cytochrome        c;    -   use of proximity assays that detect interaction between a        polypeptide having acetylation or deacetylation activity and a        substrate, e.g., cytochrome c, or fragments thereof, for        example, fluorescence proximity assays.    -   use of a two hybrid assay to detect interaction between a        polypeptide having acetylation or deacetylation activity and        cytochrome c or fragments thereof. In some instances, the two        hybrid assay can be evaluated in the presence of a test        compound, e.g., to determine if the test compound disrupts or        interferes with an interaction. Two hybrid assays can, for        example, be conducted using yeast or bacterial systems.    -   use of radio-labeled substrates, e.g. ³⁵S, ³H, ¹⁴C, e.g., to        determine acetylation status, metabolic status, rate of protein        synthesis, inter alia.    -   use of antibodies specific for certain acetylated or        de-acetylated forms of the substrate.

Various screening assays are described in more detail below.

Any assay herein, e.g., an in vitro assay or an in vivo assay, can beperformed individually, e.g., just with the test compound, or withappropriate controls. For example, a parallel assay without the testcompound, or other parallel assays without other reaction components,e.g., without a target or without a substrate. Alternatively, it ispossible to compare assay results to a reference, e.g., a referencevalue, e.g., obtained from the literature, a prior assay, and so forth.Appropriate correlations and art known statistical methods can be usedto evaluate an assay result.

A “compound” or “test compound” can be any chemical compound, forexample, a macromolecule (e.g., a polypeptide, a protein complex, or anucleic acid) or a small molecule (e.g., an amino acid, a nucleotide, anorganic or inorganic compound). The test compound can have a formulaweight of less than about 10,000 grams per mole, less than 5,000 gramsper mole, less than 1,000 grams per mole, or less than about 500 gramsper mole. The test compound can be naturally occurring (e.g., a herb ora nature product), synthetic, or both. Examples of macromolecules areproteins, protein complexes, and glycoproteins, nucleic acids, e.g.,DNA, RNA and PNA (peptide nucleic acid). Examples of small molecules arepeptides, peptidomimetics (e.g., peptoids), amino acids, amino acidanalogs, polynucleotides, polynucleotide analogs, nucleotides,nucleotide analogs, organic or inorganic compounds e.g., heteroorganicor organometallic compounds. A test compound can be the only substanceassayed by the method described herein. Alternatively, a collection oftest compounds can be assayed either consecutively or concurrently bythe methods described herein.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic compounds (potential modulator or ligandcompounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT PublicationNo. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomerssuch as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN,January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.5,288,514, and the like). Additional examples of methods for thesynthesis of molecular libraries can be found in the art, for examplein: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb etal. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckemmann et al.(1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303;Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al.(1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J.Med. Chem. 37:1233.

Some exemplary libraries are used to generate variants from a particularlead compound. One method includes generating a combinatorial library inwhich one or more functional groups of the lead compound are varied,e.g., by derivatization. Thus, the combinatorial library can include aclass of compounds which have a common structural feature (e.g.,framework).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N. J., Asinex, Moscow, Ru, Tripos,Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals,Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

The test compounds of the present invention can also be obtained from:biological libraries; peptoid libraries (libraries of molecules havingthe functionalities of peptides, but with a novel, non-peptide backbonewhich are resistant to enzymatic degradation but which neverthelessremain bioactive; see, e.g., Zuckermann, R. N. et al. (1994) J. Med.Chem. 37:2678-85); spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibraries include libraries of nucleic acids and libraries of proteins.Some nucleic acid libraries encode a diverse set of proteins (e.g.,natural and artificial proteins; others provide, for example, functionalRNA and DNA molecules such as nucleic acid aptamers or ribozymes. Apeptoid library can be made to include structures similar to a peptidelibrary. (See also Lam (1997) Anticancer Drug Des. 12:145). A library ofproteins may be produced by an expression library or a display library(e.g., a phage display library).

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner,U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409),plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or onphage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382;Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).

In Vitro Assays

In some embodiments, interaction with, e.g., binding of, a polypeptidehaving acetylation or deacetylation activity, can be assayed in vitro.The reaction mixture can include a co-factor of the polypeptide such asNAD and/or a NAD analog.

In other embodiments, the reaction mixture can include a cytochrome c,and compounds can be screened, e.g., in an in vitro assay, to evaluatethe ability of a test compound to modulate interaction between thepolypeptide and a substrate, e.g., a cytochrome c. This type of assaycan be accomplished, for example, by coupling one of the components,with a radioisotope or enzymatic label such that binding of the labeledcomponent to the other can be determined by detecting the labeledcompound in a complex. A component can be labeled with ¹²⁵I, ³⁵S, ¹⁴C,or ³H, either directly or indirectly, and the radioisotope detected bydirect counting of radioemmission or by scintillation counting.Alternatively, a component can be enzymatically labeled with, forexample, horseradish peroxidase, alkaline phosphatase, or luciferase,and the enzymatic label detected by determination of conversion of anappropriate substrate to product. Competition assays can also be used toevaluate a physical interaction between a test compound and a target.

Cell-free assays involve preparing a reaction mixture of the targetprotein (e.g., a SIR polypeptide) and the test compound under conditionsand for a time sufficient to allow the two components to interact andbind, thus forming a complex that can be removed and/or detected.

The interaction between two molecules can also be detected, e.g., usinga fluorescence assay in which at least one molecule is fluorescentlylabeled. One example of such an assay includes fluorescence energytransfer (FET or FRET for fluorescence resonance energy transfer) (see,for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos,et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first,‘donor’ molecule is selected such that its emitted fluorescent energywill be absorbed by a fluorescent label on a second, ‘acceptor’molecule, which in turn is able to fluoresce due to the absorbed energy.Alternately, the ‘donor’ protein molecule may simply utilize the naturalfluorescent energy of tryptophan residues. Labels are chosen that emitdifferent wavelengths of light, such that the ‘acceptor’ molecule labelmay be differentiated from that of the ‘donor’. Since the efficiency ofenergy transfer between the labels is related to the distance separatingthe molecules, the spatial relationship between the molecules can beassessed. In a situation in which binding occurs between the molecules,the fluorescent emission of the ‘acceptor’ molecule label in the assayshould be maximal. A FET binding event can be conveniently measuredthrough standard fluorometric detection means well known in the art(e.g., using a fluorimeter).

Another example of a fluorescence assay is fluorescence polarization(FP). For FP, only one component needs to be labeled. A bindinginteraction is detected by a change in molecular size of the labeledcomponent. The size change alters the tumbling rate of the component insolution and is detected as a change in FP. See, e.g., Nasir et al.(1999) Comb Chem HTS 2:177-190; Jameson et al. (1995) Methods Enzymol246:283; Seethala et al. (1998) Anal Biochem. 255:257. Fluorescencepolarization can be monitored in multiwell plates, e.g., using the TecanPolarion™ reader. See, e.g., Parker et al. (2000) Journal ofBiomolecular Screening 5:77-88; and Shoeman, et al. (1999) 38,16802-16809.

In another embodiment, determining the ability of the protein to bind toa target molecule can be accomplished using real-time BiomolecularInteraction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C.(1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin.Struct. Biol. 5:699-705). “Surface plasmon resonance” or “BIA” detectsbiospecific interactions in real time, without labeling any of theinteractants (e.g., BIAcore). Changes in the mass at the binding surface(indicative of a binding event) result in alterations of the refractiveindex of light near the surface (the optical phenomenon of surfaceplasmon resonance (SPR)), resulting in a detectable signal which can beused as an indication of real-time reactions between biologicalmolecules.

In one embodiment, a polypeptide, e.g., a SIR polypeptide, is anchoredonto a solid phase. The polypeptide/test compound complexes anchored onthe solid phase can be detected at the end of the reaction, e.g., thebinding reaction. For example, a SIR polypeptide can be anchored onto asolid surface, and the test compound, (which is not anchored), can belabeled, either directly or indirectly, with detectable labels discussedherein.

It may be desirable to immobilize either the polypeptide or an antibodyto the polypeptide to facilitate separation of complexed fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a test compound to apolypeptide, or interaction of a protein with a second component in thepresence and absence of a candidate compound, can be accomplished in anyvessel suitable for containing the reactants. Examples of such vesselsinclude microtiter plates, test tubes, and micro-centrifuge tubes. Inone embodiment, a fusion protein can be provided which adds a domainthat allows one or both of the proteins to be bound to a matrix. Forexample, glutathione-S-transferase/SIR polypeptide fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or a SIR protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level ofpolypeptide binding or activity determined using standard techniques.

Other techniques for immobilizing either a protein or a target moleculeon matrices include using conjugation of biotin and streptavidin.Biotinylated protein or target molecules can be prepared frombiotin-NHS(N-hydroxy-succinimide) using techniques known in the art(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical).

In order to conduct the assay, the non-immobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynon-immobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed: Wherethe previously non-immobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface, e.g.,using a labeled antibody specific for the immobilized component (theantibody, in turn, can be directly labeled or indirectly labeled with,e.g., a labeled anti-Ig antibody).

In one embodiment, this assay is performed utilizing antibodies reactivewith a protein or target molecules but which do not interfere withbinding of the protein to its target molecule. Such antibodies can bederivatized to the wells of the plate, and unbound target or the proteintrapped in the wells by antibody conjugation. Methods for detecting suchcomplexes, in addition to those described above for the GST-immobilizedcomplexes, include immunodetection of complexes using antibodiesreactive with the protein or target molecule, as well as enzyme-linkedassays which rely on detecting an enzymatic activity associated with theprotein or target molecule.

Alternatively, cell free assays can be conducted in a liquid phase. Insuch an assay, the reaction products are separated from unreactedcomponents, by any of a number of standard techniques, including but notlimited to: differential centrifugation (see, for example, Rivas, G.,and Minton, A. P., (1993) Trends Biochem Sci 18:284-7); chromatography(gel filtration chromatography, ion-exchange chromatography);electrophoresis (see, e.g., Ausubel, F. et al., eds. Current Protocolsin Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation(see, for example, Ausubel, F. et al., eds. (1999) Current Protocols inMolecular Biology, J. Wiley: New York). Such resins and chromatographictechniques are known to one skilled in the art (see, e.g., Heegaard, N.H., (1998) J Mol Recognit 11:141-8; Hage, D. S., and Tweed, S. A. (1997)J Chromatogr B Biomed Sci Appl. 699:499-525). Further, fluorescenceenergy transfer may also be conveniently utilized, as described herein,to detect binding without further purification of the complex fromsolution.

In a preferred embodiment, the assay includes contacting the protein orbiologically active portion thereof with a known compound which binds aprotein to form an assay mixture, contacting the assay mixture with atest compound, and determining the ability of the test compound tointeract with a protein, wherein determining the ability of the testcompound to interact with the protein includes determining the abilityof the test compound to preferentially bind to the protein orbiologically active portion thereof, or to modulate the activity of atarget molecule such as acetylation status, as compared to the knowncompound.

The target products of the invention can, in vivo, interact with one ormore cellular or extracellular macromolecules, such as proteins. For thepurposes of this discussion, such cellular and extracellularmacromolecules are referred to herein as “binding partners.” Compoundsthat disrupt such interactions can be useful in regulating the activityof the target product. Such compounds can include, but are not limitedto molecules such as antibodies, peptides, and small molecules.

To identify compounds that interfere with the interaction between thetarget product and its binding partner(s), a reaction mixture containingthe target product and the binding partner is prepared, under conditionsand for a time sufficient, to allow the two products to form complex. Inorder to test an inhibitory agent, the reaction mixture is provided inthe presence and absence of the test compound. The test compound can beinitially included in the reaction mixture, or can be added at a timesubsequent to the addition of the target and its cellular orextracellular binding partner. Control reaction mixtures are incubatedwithout the test compound or with a placebo. The formation of anycomplexes between the target product and the cellular or extracellularbinding partner is then detected. The formation of a complex in thecontrol reaction, but not in the reaction mixture containing the testcompound, indicates that the compound interferes with the interaction ofthe target product and the interactive binding partner. Additionally,complex formation within reaction mixtures containing the test compoundand normal target product can also be compared to complex formationwithin reaction mixtures containing the test compound and mutant targetproduct. This comparison can be important in those cases wherein it isdesirable to identify compounds that disrupt interactions of mutant butnot normal target products.

These assays can be conducted in a heterogeneous or homogeneous format.Heterogeneous assays involve anchoring either the target product or thebinding partner onto a solid phase, and detecting complexes anchored onthe solid phase at the end of the reaction. In homogeneous assays, theentire reaction is carried out in a liquid phase. In either approach,the order of addition of reactants can be varied to obtain differentinformation about the compounds being tested. For example, testcompounds that interfere with the interaction between the targetproducts and the binding partners, e.g., by competition, can beidentified by conducting the reaction in the presence of the testsubstance. Alternatively, test compounds that disrupt preformedcomplexes, e.g., compounds with higher binding constants that displaceone of the components from the complex, can be tested by adding the testcompound to the reaction mixture after complexes have been formed. Thevarious formats are briefly described below.

In a heterogeneous assay system, either the target product or thepartner, is anchored onto a solid surface (e.g., a microtiter plate),while the non-anchored species is labeled, either directly orindirectly. The anchored species can be immobilized by non-covalent orcovalent attachments. Alternatively, an immobilized antibody specificfor the species to be anchored can be used to anchor the species to thesolid surface.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. Where the non-immobilized species is pre-labeled, the detectionof label immobilized on the surface indicates that complexes wereformed. Where the non-immobilized species is not pre-labeled, anindirect label can be used to detect complexes anchored on the surface;e.g., using a labeled antibody specific for the initiallynon-immobilized species (the antibody, in turn, can be directly labeledor indirectly labeled with, e.g., a labeled anti-Ig antibody). Dependingupon the order of addition of reaction components, test compounds thatinhibit complex formation or that disrupt preformed complexes can bedetected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds that inhibit complex or that disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. For example, a preformed complex of the target product and theinteractive cellular or extracellular binding partner product isprepared in that either the target products or their binding partnersare labeled, but the signal generated by the label is quenched due tocomplex formation (see, e.g., U.S. Pat. No. 4,109,496 that utilizes thisapproach for immunoassays). The addition of a test substance thatcompetes with and displaces one of the species from the preformedcomplex will result in the generation of a signal above background. Inthis way, test substances that disrupt target product-binding partnerinteraction can be identified.

Many of the screening assays described herein have discussed screeningfor molecules with regard to a SIR protein or other protein havingacetylation or deacetylation activity, however, the same assays can alsobe used to screen for molecules with regard to cytochrome c.

In yet another aspect, the polypeptide can be used as “bait proteins” ina two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No.5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J.Biol. Chem. 268.12046-12054; Bartel et al. (1993) Biotechniques14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and BrentWO94/10300), to identify other proteins, which bind to or interact witha polypeptide (“polypeptide binding partners”) and are involved inacetylation status of cytochrome c. Such a polypeptide binding partnercan be an activator or inhibitor of signals by the proteins.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a protein is fusedto a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. (Alternatively the protein can be thefused to the activator domain.) If the “bait” and the “prey” proteinsare able to interact, in vivo, forming a polypeptide-dependent complex,the DNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., lacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing thefunctional transcription factor can be isolated and used to obtain thecloned gene which encodes the protein which interacts with the protein,e.g., the protein having acetylation or deacetylation activity. Inanother embodiment, the two-hybrid assay is used to monitor aninteraction between two components, e.g., a polypeptide havingacetylation or deacetylation activity such as a SIR polypeptide and,e.g., cytochrome c, that are known to interact. The two hybrid assay isconducted in the presence of a test compound, and the assay is used todetermine whether the test compound enhances or diminishes theinteraction between the components.

In another embodiment, modulators of gene expression are identified. Forexample, a cell or cell free mixture is contacted with a candidatecompound and the expression of the mRNA or protein evaluated relative tothe level of expression of mRNA or protein in the absence of thecandidate compound. When expression of the mRNA or protein is greater inthe presence of the candidate compound than in its absence, thecandidate compound is identified as a stimulator of mRNA or proteinexpression. Alternatively, when expression of the mRNA or protein isless (statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of the mRNA or protein expression. The level of the mRNA orprotein expression can be determined by methods for detecting mRNA orproteins, e.g., using probes or antibodies, e.g., labeled probes orantibodies.

Cell-Based Assays

In another embodiment, the assay, e.g., the assay for selectingcompounds which interact with a polypeptide and/or which effect (e.g.,induce) apoptosis, can be a cell-based assay. The cell based assay caninclude contacting a cell expressing a polypeptide having acetylation ordeacetylation activity and/or cytochrome c with a test compound anddetermining the ability of the test compound to modulate (e.g. stimulateor inhibit) an activity of the polypeptide or cytochrome c, and/ordetermine the ability of the test compound to modulate polypeptideand/or cytochrome c expression, e.g., by detecting nucleic acids (e.g.,mRNA or cDNA) or proteins in the cell. A preferred activity is thedeacetylation function of a polypeptide of cytochrome c; a furtherpreferred activity is the ability to cause apoptosis. Determining theability of the test compound to modulate the activity of a polypeptidecan be accomplished, for example, by determining the ability of apolypeptide to bind to or interact with the test molecule, and bydetermining the ability of the test molecule to stimulate apoptosis.Cell-based systems can be used to identify compounds that decrease thepolypeptides expression and/or activity and/or effect, e.g., decrease orprevent apoptosis, or visa versa. Such cells can be recombinant ornon-recombinant, such as cell lines that express the gene encoding thepolypeptide and/or the cytochrome c gene. In some embodiments, the cellscan be recombinant or non-recombinant cells which express atranscription factor. Preferred systems are mammalian or yeast cellsthat express a polypeptide having acetylation or deacetylation activityand cytochrome c. In utilizing such systems, cells are exposed tocompounds suspected of decreasing expression of a deacetylatingpolypeptide and/or decreasing deacetylation activity of the polypeptideand/or reducing apoptosis, or compounds suspected of increasingexpression of a deacetylating polypeptide and/or increasing adeacetylation activity and/or inducing apoptosis. After exposure, thecells are assayed, for example, for expression of the gene or activityof the protein. Alternatively, the cells may also be assayed for theinhibition of the deacetylation function of a polypeptide, or theapoptotic or cytostatic function. In one embodiment, the visualassessment can be used for evidence of apoptosis, e.g., nuclearfragmentation.

Another preferred cell for a cell-based assay comprises a yeast celltransformed with a vector comprising the Sir2 gene, a homolog of human aSIRT1. One use for a yeast cell expressing Sir2 is to mutagenize theyeast and screen for yeast that will survive only when the Sir2polypeptide is functioning normally. Synthetic lethal screens aredescribed in Holtzman et al. (1993), J. Cell Bio. 122: 635-644. Theyeast that require Sir2 function for survival can then be used to screentest compounds for those that inhibit Sir2 activity. Test compounds thatresults in a decrease in yeast survival are likely inhibitors of Sir2 inthis system.

A cell used in the methods of the invention can be from a stable cellline or a primary culture obtained from an organism, e.g., a organismtreated with the test compound.

In addition to cell-based and in vitro assay systems, non-humanorganisms, e.g., transgenic non-human organisms, can also be used. Atransgenic organism is one in which a heterologous DNA sequence ischromosomally integrated into the germ cells of the animal. A transgenicorganism will also have the transgene integrated into the chromosomes ofits somatic cells. Organisms of any species, including, but not limitedto: yeast, worms, flies, fish, reptiles, birds, mammals (e.g., mice,rats, rabbits, guinea pigs, pigs, micro-pigs, and goats), and non-humanprimates (e.g., baboons, monkeys, chimpanzees) may be used in themethods of the invention.

A transgenic cell or animal used in the methods of the invention caninclude a transgene that encodes, e.g., a copy of a polypeptide havingacetylation or deacetylation activity and/or cytochrome c, e.g., apolypeptide having acetylation or deacetylation activity or cytochrome cpolypeptide that was evaluated for an interaction with the testcompound. The transgene can encode a protein that is normally exogenousto the transgenic cell or animal, including a human protein, e.g., ahuman SIR polypeptide and/or human cytochrome c. The transgene can belinked to a heterologous or a native promoter. Methods of makingtransgenic cells and animals are known in the art.

Accordingly, in another embodiment, the invention features a method ofidentifying a compound as a candidate of treatment of unwanted cellproliferation, e.g., cancer treatment. The method includes: providing acompound which interacts with, e.g., binds to, a SIR polypeptide;evaluating the effect of the compound on apoptosis, wherein a compoundthat results in apoptosis is subjected to further evaluation steps; andfurther evaluating the effect of the test compound on a subject, e.g.,an animal model, e.g., an animal model for cancer.

The interaction between a test compound and the polypeptide havingacetylation or deacetylation activity, e.g., a SIR polypeptide, can beperformed by any of the methods described herein, e.g., using cell-basedassays or cell-free in vitro assays.

Cytochrome c can be detected using a monoclonal antibody (clone 7H, 8.2;C12, PharMingen, San Diego, Calif.),

Structural Activity Relationships

It is also possible to use structure-activity relationships (SAR) andstructure-based design principles to find compounds that have improvedeffects on a polypeptide having acetylation or deacetylation activity.SARs provide information about the activity of related compounds in atleast one relevant assay. Correlations are made between structuralfeatures of a compound of interest and an activity. For example, it maybe possible by evaluating SARs for a family of compounds that interactwith a polypeptide to identify one or more structural features requiredfor activity. A library of compounds can then be produced that varythese features, and then the library is screened. Structure-based designcan include determining a structural model of the physical interactionof the compound and its target. The structural model can indicate how anantagonist of the target can be engineered. Such antagonist may beuseful in altering lifespan regulation.

Both the SAR and the structure-based design approach can be used toidentify a pharmacophore. Pharmacophores are a highly valuable anduseful concept in drug discovery and drug-lead optimization. Apharmacophore is defined as a distinct three dimensional (3D)arrangement of chemical groups essential for biological activity. Sincea pharmaceutically active molecule must interact with one or moremolecular structures within the body of the subject in order to beeffective, and the desired functional properties of the molecule arederived from these interactions, each active compound must contain adistinct arrangement of chemical groups which enable this interaction tooccur. The chemical groups, commonly termed descriptor centers, can berepresented by (a) an atom or group of atoms; (b) pseudo-atoms, forexample a center of a ring, or the center of mass of a molecule; (c)vectors, for example atomic pairs, electron lone pair directions, or thenormal to a plane. Once formulated a pharmacophore can be used to searcha database of chemical compound, e.g., for those having a structurecompatible with the pharmacophore. See, for example, U.S. Pat. No.6,343,257; Y C. Martin, 3D Database Searching in Drug Design, J. Med.Chem. 35, 2145 (1992); and A. C. Good and J. S. Mason, Three DimensionalStructure Database Searches, Reviews in Comp. Chem. 7, 67 (1996).Database search queries are based not only on chemical propertyinformation but also on precise geometric information.

Computer-based approaches can use database searching to find matchingtemplates; Y C. Martin, Database searching in drug design, J. MedicinalChemistry, vol. 35, pp 2145-54 (1992), which is herein incorporated byreference. Existing methods for searching 2-D and 3-D databases ofcompounds are applicable. Lederle of American Cyanamid (Pearl River,N.Y.) has pioneered molecular shape-searching, 3D searching andtrend-vectors of databases. Commercial vendors and other research groupsalso provide searching capabilities (MACSS-3D, Molecular Design Ltd.(San Leandro, Calif.); CAVEAT, Lauri, G et al., University of California(Berkeley, Calif.); CHEM-X, Chemical Design, Inc. (Mahwah, N.J.)).Software for these searches can be used to analyze databases ofpotential drug compounds indexed by their significant chemical andgeometric structure (e.g., the Standard Drugs File (Derwent PublicationsLtd., London, England), the Bielstein database (Bielstein Information,Frankfurt, Germany or Chicago), and the Chemical Registry database (CAS,Columbus, Ohio)).

Once a compound is identified that matches the pharmocophore, it can betested for activity, e.g., for binding to a component of a polypeptideand/or for a biological activity, e.g., deacetylation and/or apoptosis.See, e.g., the Screening Methods described above.

Organismal Assays

Still other methods for evaluating a test compound include organismalbased assays, e.g., using a mammal (e.g., a mouse, rat, primate, orother non-human), or other animal (e.g., Xenopus, zebrafish, or aninvertebrate such as a fly or nematode). In some cases, the organism isa transgenic organism, e.g., an organism which includes a heterologousSIR and/or cytochrome c component, (e.g., from a mammal, e.g., a human).The test compound can be administered to the organism once or as aregimen (regular or irregular). A parameter of the organism is thenevaluated, e.g., an age-associated parameter or a parameter of thecytochrome c-mediated apoptosis pathway. Test compounds that areindicated as of interest result in a change in the parameter relative toa reference, e.g., a parameter of a control organism. Other parameters(e.g., related to toxicity, clearance, and pharmacokinetics) can also beevaluated.

In some embodiment, the test compound is evaluated using an animal thathas a particular disorder, e.g., an age associated disorder. Thesedisorders provide a sensitized system in which the test compound'seffects on physiology can be observed. Exemplary disorders include:denervation, disuse atrophy; metabolic disorders (e.g., disorder ofobese and/or diabetic animals such as db/db mouse and ob/ob mouse);cerebral, liver ischemia; cisplatin/taxol/vincristine models; varioustissue (xenograph) transplants; transgenic bone models; Pain syndromes(include inflammatory and neuropathic disorders); Paraquot, genotoxic,oxidative stress models; pulmonary obstruction (e.g., asthma models);and tumor models. In a preferred embodiment, the animal model is ananimal that has an altered phenotype when calorically restricted. Forexample, F344 rats provide a useful assay system for evaluating a testcompound. When calorically restricted, F344 rats have a 0 to 10%incidence of nephropathy. However, when fed ad libitum, they have a 60to 100% incidence of nephropathy. See Table 2. TABLE 2 F344 rats -Frequency of nephropathy. Months Ad lib CR 6  0% 0% 12  60% 0% 18 100%0% 24 100% 0%

Additional animals are listed in Table 2: TABLE 3 Mean Lifespan (months)Model Ad lib CR Predisposition SH Rat 18 30 Hypertension SA Mouse 10 15Amyloid NZB Mouse 12 16 SLE kd/kd Mouse 8 18 Nephritis MRL/1 Mouse 6 >15Autoimmune ob/ob Mouse 14 26 Diabetes

To evaluate a test compound, it is administered to the animal (e.g., anF344 rat or an animal listed in Table 3), and a parameter of the animalis evaluated, e.g., after a period of time. The animal can be fed adlibitum or normally (e.g., not under caloric restriction, although someparameters can be evaluated under such conditions). Typically, a cohortof such animals is used for the assay. Generally, a test compound can beindicated as favorably altering lifespan regulation in the animal if thetest compound affects the parameter in the direction of the phenotype ofa similar animal subject to caloric restriction. Such test compounds maycause at least some of the lifespan regulatory effects of caloricrestriction, e.g., a subset of such effects, without having to deprivethe organism of caloric intake.

In one embodiment, the parameter is an age-associated or diseaseassociated parameter, e.g., a symptom of the disorder associated withthe animal model (e.g., the disorder in the “Predisposition column ofTable 3). For example, the test compound can be administered to the SHRat, and blood pressure is monitored. A test compound that is favorablyindicated can cause an amelioration of the symptom relative to a similarreference animal not treated with the compound.

Still other in vivo models and organismal assays include:

-   -   insulin sensitivity;    -   Tumors: spontaneous, induced, grafts, cytochrome c−, cytochrome        c+;    -   Autoimmune: NZB mice;    -   Cognition: learning & memory models;    -   Bone disease: ovariectomy osteoporosis model;    -   Joint disease: adjuvant arthritis;    -   Kidney disease: kd/kd mice;    -   Diabetes & complications: streptozotocin model; and    -   Canine stroke & ischemia models.

In assessing whether a test compound is capable of inhibiting thecytochrome c-mediated apoptosis pathway for the purpose of altering lifespan regulation, a number of age-associated parameters or biomarkers canbe monitored or evaluated. Exemplary age associated parameters include:(i) lifespan of the cell or the organism; (ii) presence or abundance ofa gene transcript or gene product in the cell or organism that has abiological age-dependent expression pattern; (iii) resistance of thecell or organism to stress; (iv) one or more metabolic parameters of thecell or organism; (v) proliferative capacity of the cell or a set ofcells present in the organism; and (vi) physical appearance or behaviorof the cell or organism.

Characterization of molecular differences between two such organisms,e.g., one reference organism and one organism treated with an cytochromec-mediated apoptosis modulator can reveal a difference in thephysiological state of the organisms. The reference organism and thetreated organism are typically the same chronological age. Generally,organisms of the same chronological age may have lived for an amount oftime within 15, 10, 5, 3, 2 or 1% of the average lifespan of a wild typeorganism of that species. In a preferred embodiment, the organisms areadult organisms, e.g. the organisms have lived for at least an amount oftime in which the average wild type organism has matured to an age atwhich it is competent to reproduce.

In some embodiments, the organismal screening assay is performed beforethe organisms exhibit overt physical features of aging. For example, theorganisms may be adults that have lived only 10, 30, 40, 50, 60, or 70%of the average lifespan of a wild type organism of the same species.

Age-associated changes in metabolism, immune competence, and chromosomalstructure have been reported. Any of these changes can be evaluated,either in a test subject (e.g., for an organism based assay), or for apatient (e.g., prior, during or after treatment with a therapeuticdescribed herein.

In another embodiment, a marker associated with caloric restriction isevaluated in a subject organism of a screening assay (or a treatedsubject). Although these markers may not be age-associated, they may beindicative of a physiological state that is altered when the cytochromec-mediated apoptosis pathway is modulated. The marker can be an mRNA orprotein whose abundance changes in calorically restricted animals. WO01/12851 and U.S. Pat. No. 6,406,853 describe exemplary markers.

In a related aspect, the invention features a method of evaluating atest compound using a plurality of biomarkers. This can be done byprofiling the sample. The method includes providing a cell or organismand a test compound; contacting the test compound to the cell; obtaininga subject expression profile for the contacted cell; and comparing thesubject expression profile to one or more reference profiles. Theprofiles include a value representing the level of expression ofmolecules previously determined to be correlated with SIR activityand/or the cytochrome c-mediated apoptosis (see, e.g., below). In apreferred embodiment, the subject expression profile is compared to atarget profile, e.g., a profile for a normal cell or for desiredcondition of a cell. The test compound is evaluated favorably if thesubject expression profile is more similar to the target profile than anexpression profile obtained from an uncontacted cell.

Similarity of profiles can be determined by a variety of metric,including Euclidean distance in a n-dimensional space, where n is thenumber of different values within the profile. Other metrics, forexample, include weighting factors that basis different values accordingto their importance for the comparison.

Profiles, e.g., profiles obtained from nucleic acid array or proteinarrays can be used to compare samples and/or cells in a variety ofstates as described in Golub et al. ((1999) Science 286:531). In oneembodiment, multiple expression profiles from different conditions andincluding replicates or like samples from similar conditions arecompared to identify nucleic acids whose expression level is predictiveof the sample and/or condition. Each candidate nucleic acid can be givena weighted “voting” factor dependent on the degree of correlation of thenucleic acid's expression and the sample identity. A correlation can bemeasured using a Euclidean distance or the Pearson correlationcoefficient.

Combinatorial Systems

The organisms described herein may be deficient in the activity of anyprotein that is associated with aging, e.g., associated with theregulation of lifespan. Some exemplary genes and homologs of genes whichencode proteins that are associated with the regulation of lifespan arelisted in Table 1. For example, mutant or otherwise altered (e.g., RNAitreated or transgenic) organisms that are altered for SIR activity or acytochrome c-apoptosis activity can also include an alteration in acomponent that is listed in Table 1 or that directly interacts with acomponent in Table 1.

Other types of combinatorial systems include environmental treatment ofan organism that is mutant or otherwise altered (e.g., RNAi treated ortransgenic) with respect to SIR polypeptide activity and/or cytochromec-mediated apoptotic activity. Exemplary environmental treatmentsinclude stress (e.g., oxidative stress, genotoxic stress, H₂O₂, heavymetal exposure), caloric restriction, and treatment with a drug, e.g., ahistone deacetylase inhibitor. TABLE 1 Organism Gene name DescriptionExemplary homologs S. cerevisiae SIR2 NAD-dependent Murine Sir2alphahistone (GenBank AccNo: deacetylase AF214646), human A SIR POLYPEPTIDE(GenBank Acc No: AF083106) human Sir2 SIRT3 GenBank Accession No:AF083108; human Sir2 SIRT4 GenBank Accession No: AF083109; human Sir2SIRT5 GenBank Accession No: AF083110 SIR3 Regulator of chromatinsilencing SIR4 Regulator of chromatin silencing RPD3 Histone deacetylaseFOB1 Suppresses rDNA replication SGS1 Werners-like DNA helicase SNF1Kinase involved in carbon source utilization SIP2 SNF1 co-repressor SNF4SNF1 co-activator NPT1 Involved in NAD synthesis RTG2 Sensor ofmitochondrial disfunction Coq7 Regulator of ubiquinone synthesis C.elegans Daf-2 Insulin/IGF-1 insulin or IGF receptor homolog receptorAge-1 PI(3) kinase PI(3) kinase Pdk-1 PDK-1 Daf-18 Phosphatase PTENDaf-16 Forkhead/winged- AFX, FKHR, FKHRL1 helix family transcriptionfactor Ceinsulin-1 Insulin/IGF-1-like insulin or IGF homolog moleculesCtl-1 Cytosolic catalase MEV-1 Cytochrome B Cytochrome B subunit ofsubunit of mitochondrial mitochondrial succinate succinate dehydrogenasedehydrogenase Sod-3 Mn-superoxide superoxide dismutase dismutase Clk-1Regulator of ubiquinone synthesis [Eat mutants] Tkr-1 Tyrosine kinaseSpe-10 Unknown (sperm defective) Spe-26 Unknown (sperm defective) Old-1Receptor tyrosine kinase Kin-29 Serine Threonine Kinase Drosophila IndyCarboxylate hNaDC-1, accession GenBank transporter No. U26209, SDCT2,accession accession no. no. AF081825, NaDC-1, AE003519 accession no.U12186, mNaDC-1, accession no. AF 201903, human solute carrier family13, member 2 GenBank NP_003975.1, human sodium-dependent high-affinitydicarboxylate transporter 3, human carrier family 13 (sodium/sulfatesymporters), member 1, human hypothetical protein XP_091606, humancarrier family 13 (sodium/sulfate symporters) member 4 (GenBankNP_036582), Cu/Zn-SOD superoxide dismutase Methuselah Putative G-protein-coupled 7 transmembrane domain receptor Mus p66shc Signalingadaptor musculus PROP1 Homeodomain protein Growth hormone Growth hormoneReleasing hormone receptorRNAI

It is also possible to regulate activity of a polypeptide havingacetylation or deacetylation activity and/or cytochrome c-mediatedapoptotic activity using a double-stranded RNA (dsRNA) that mediates RNAinterference (RNAi). The dsRNA can be delivered to cells or to anorganism. Endogenous components of the cell or organism can trigger RNAinterference (RNAi) which silences expression of genes that include thetarget sequence. dsRNA can be produced by transcribing a cassette inboth directions, for example, by including a T7 promoter on either sideof the cassette. The insert in the cassette is selected so that itincludes a sequence complementary to a nucleic acid encoding a componentof the cytochrome c-mediate apoptosis pathway. The sequence need not befull length, for example, an exon, or at least 50 nucleotides. Thesequence can be from the 5′ half of the transcript, e.g., within 1000,600, 400, or 300 nucleotides of the ATG See also, the HiScribe™ RNAiTranscription Kit (New England Biolabs, MA) and Fire, A. (1999) TrendsGenet. 15, 358-363.

dsRNA can be digested into smaller fragments. See, e.g., U.S. patentapplication 2002-0086356 and 2003-0084471. In one embodiment, an siRNAis used. siRNAs are small double stranded RNAs (dsRNAs) that optionallyinclude overhangs. For example, the duplex region is about 18 to 25nucleotides in length, e.g., about 19, 20, 21, 22, 23, or 24 nucleotidesin length. Typically the siRNA sequences are exactly complementary tothe target mRNA.

dsRNAs (and siRNA's in particular) can be used to silence geneexpression in mammalian cells. See, e.g., Clemens, J. C. et al. (2000)Proc. Natl. Sci. USA 97, 6499-6503; Billy, E. et al. (2001) Proc. Natl.Sci. USA 98, 14428-14433; Elbashir et al. (2001) Nature.411(6836):494-8; Yang, D. et al. (2002) Proc. Natl. Acad. Sci. USA 99,9942-9947.

dsRNA molecules can be used to provide cells and organisms (e.g.,mammalian cells and organisms, and nematode mammalian cells andorganisms) that are deficient in a deacetylation, acetylation, and/orcytochrome c-mediated apoptotic activity. Such cells and organisms areuseful tools for evaluating heterologous molecules and test compoundsfor activity, e.g., an activity that modulates lifespan regulation.

Pharmaceutical Compositions

A compound that modulates the cytochrome c-mediated apoptotic pathway,e.g., modulates expression and/or activity of a polypeptide havingacetylation or deacetylation activity and/or cytochrome c can beincorporated into a pharmaceutical composition for administration to asubject, e.g., a human, a non-human animal, e.g., an animal patient(e.g., pet or agricultural animal) or an animal model (e.g., an animalmodel for aging or a metabolic disorder (e.g., a pancreatic or insulinrelated disorder). Such compositions typically include a small molecule(e.g., a small molecule that is a SIR activator, or a SIR inactivator),nucleic acid molecule, protein, or antibody and a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” includes solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Other active compounds can also be incorporated into the compositions.

Exemplary compounds that can be used for inducing acetylation ofcytochrome c and/or decreasing apoptosis can include antisense to adeacetylating polypeptide, RNAi, an antibody, an intrabody, and othercompounds identified by a method described herein, e.g., compounds thatreduce apoptosis in a deacetylating polypeptide and cytochrome cexpressing cell. Exemplary compounds that can be used for reducingacetylation of cytochrome c and/or increasing apoptosis can include apurified deacetylating polypeptide, expression of a deacetylatingpolypeptide from heterologous genes, or by increasing the expression ofendogenous sequence encoding a deacetylating polypeptide and othercompounds identified by a method described herein, e.g., compounds thatinduces apoptosis in a deacetylating polypeptide and cytochrome cexpressing cell.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (ASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fingi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art. Thecompounds can also be prepared in the form of suppositories or retentionenemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to particular cells, e.g., a pituitarycell) can also be used as pharmaceutically acceptable carriers. Thesecan be prepared according to methods known to those skilled in the art,for example, as described in U.S. Pat. No. 4,522,811.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The protein or polypeptide can be administered onetime per week for between about 1 to 10 weeks, preferably between 2 to 8weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. The skilled artisan willappreciate that certain factors may influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health and/or age of the subject, and other diseases present.Moreover, treatment of a subject with a therapeutically effective amountof a compound can include a single treatment or, preferably, can includea series of treatments.

For antibody compounds that modulate SIR components, one preferreddosage is 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg).Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration. A method forlipidation of antibodies is described by Cruikshank et al. ((1997) J.Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193).

The present invention encompasses agents that modulate acetylationstatus of cytochrome c and the cytochrome c-mediated apoptotic pathway.An agent may, for example, be a small molecule. For example, such smallmolecules include, but are not limited to, peptides, peptidomimetics(e.g., peptoids), amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic orinorganic compounds (i.e., including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds.

Exemplary doses include milligram or microgram amounts of the smallmolecule per kilogram of subject or sample weight (e.g., about 1microgram per-kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram. It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. When one or more of these small molecules isto be administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

The nucleic acid molecules that modulate expression of a polypeptidehaving acetylation or deacetylation activity and or activity thepolypeptide and/or the cytochrome c-mediated apoptotic pathway can beinserted into vectors and used as gene therapy vectors. For example, thenucleic acid can encode a SIR polypeptide. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. Proc. Natl. Acad. Sci. USA91:3054-3057, 1994). The pharmaceutical preparation of the gene therapyvector can include the gene therapy vector in an acceptable diluent, orcan comprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Further, a variety of techniques may be utilized to modulate (e.g.,increase or inhibit) the expression, synthesis, or activity of targetgenes and/or proteins. Such molecules may include, but are not limitedto small organic molecules, peptides, purified polypeptide, sequencesencoding the polypeptides, or molecules which increase the expression ofendogenous sequence encoding the polypeptide, or other deacetylationagonists described herein.

Modulating Lifespan Regulation in Subjects

Agents that alter acetylation status of cytochrome c and the cytochromec-mediated apoptotic activity can be used to modulate lifespanregulation in subjects, e.g., animal (e.g., mammalian, e.g., humansubjects). The compositions can be administered to a subject, e.g., anadult subject, e.g., a healthy adult subject or a subject having anage-related disease. In the latter case, the method can includeevaluating a subject, e.g., to characterize a symptom of an age-relateddisease or other disease marker, and thereby identifying a subject ashaving an age-related disease or being pre-disposed to such a disease.Exemplary age-related diseases include: cancer (e.g., breast cancer,colorectal cancer, CCL, CML, prostate cancer); skeletal muscle atrophy;adult-onset diabetes; diabetic nephropathy, neuropathy (e.g., sensoryneuropathy, autonomic neuropathy, motor neuropathy, retinopathy);obesity; bone resorption; age-related macular degeneration, AIDS relateddementia, ALS, Alzheimer's, Bell's Palsy, atherosclerosis, cardiacdiseases (e.g., cardiac dysrhythmias, chronic congestive heart failure,ischemic stroke, coronary artery disease and cardiomyopathy), chronicrenal failure, type 2 diabetes, ulceration, cataract, presbiopia,glomerulonephritis, Guillan-Barre syndrome, hemorrhagic stroke,rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis,SLE, Crohn's disease, osteoarthritis, Parkinson's disease, pneumonia,Touret's syndrome, and urinary incontinence. Symptoms and diagnosis ofsuch diseases are well known to medical practitioners. The compositionsmay also be administered to individuals being treated by other means forsuch diseases, for example, individuals being treated with achemotherapeutic (e.g., and having neutropenia, atrophy, cachexia,nephropathy, neuropathy) or an elective surgery.

Subjects can be diagnosed and evaluated, e.g., before, during, and aftertreatment. Standard medical procedures can be used to monitor the healthand fitness of the subject. In addition, a parameter of metabolicactivity (e.g., insulin levels) can be monitored.

In some embodiments, the cytochrome c-mediated apoptosis modulatingagent is directed to a particular cell (e.g., by using a targetingvehicle or by using a cell-type specific regulatory sequence for anucleic acid). For example, the agent can be targeting to an adipose,liver, pancreatic, brain, or skeletal muscle cell. In some examples, thetargeted tissue participates in metabolic regulation.

Treatment of Unwanted Cell Proliferation in Subjects

Cell proliferation can be modulated to decrease, inhibit or prevent cellproliferation in disorders characterized by unwanted cell proliferationsuch as cancers. As described herein, deacetylating polypeptidesinteract with human cytochrome c protein to deacetylate cytochrome c.While not wishing to be bound by theory, a functional consequence ofthis deacetylation can be an increase of the cytochrome c's interactionwith Apaf-1 and its apoptotic activity. Stress on a cell can result inrelease of cytochrome c from the mitochondria into the cytosol where inits non-acetylated form, it can interact with Apaf-1. Interaction ofcytochrome c with Apaf-1 induces events (e.g., the activation ofcaspases) which can eventually result in apoptosis of the cell.Deacetylating cytochrome c, may enhance cytochrome c's ability tointeract with Apaf-1 and induce cytochrome c-mediated apoptoticresponse. The formation of tumors is a multistep process requiringprogressive accumulation of genetic alterations. Thus, cytochrome crelease from the mitochondria due to stress, e.g., oncogenic stress, canplay an important role in cancer by inducing apoptosis of damaged cells.A consequence of loss of pro apoptotic activity is the accumulation ofthe half-dozen or so mutations necessary for a cell to becomecarcinogenic. A second consequence may be uncontrolled cell growth,e.g., metastases, of the cell. In order to enhance or increasecytochrome c mediated apoptosis in these damaged cells, deacetylation,of cytochrome c is, preferably, reduced, inhibited or prevented.

Accordingly, in some aspect, the invention features methods of treatingunwanted cell growth, e.g., in a subject having or at risk for cancer.The method includes administering a deacetylation agonist, e.g., a SIRagonist.

As used herein, the term “cancer” is meant to include all types ofcancerous growths or oncogenic processes, metastatic tissues ormalignantly transformed cells, tissues, or organs, irrespective ofhistopathologic type or stage of invasiveness. The cancer may be amalignant or non-malignant cancer. In some embodiments, the methodsprevent or treat tumor proliferation and/or metastasis. Cancers ortumors include, but are not limited, to biliary tract cancer; braincancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer;endometrial cancer; esophageal cancer; gastric cancer; intraepithelialneoplasms; leukemias, lymphomas; liver cancer; lung cancer (e.g. smallcell and non-small cell); melanoma; neuroblastomas; oral cancer; ovariancancer; pancreatic cancer; prostate cancer; rectal cancer; sarcomas;skin cancer; testicular cancer; thyroid cancer; and renal cancer, aswell as other carcinomas and sarcomas.

“Subject,” as used herein, refers to human and non-human animals. Theterm “non-human animals” of the invention includes all vertebrates,e.g., mammals, such as non-human primates (particularly higherprimates), sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat,pig, cat, rabbits, cow, and non-mammals, such as chickens, amphibians,reptiles, etc. In a preferred embodiment, the subject is a human. Inanother embodiment, the subject is an experimental animal or animalsuitable as a disease model.

In a preferred embodiment, the method includes administering adeacetylation agonist, e.g., a SIR agonist, in combination with one ormore additional therapeutic agent or agents, e.g., a therapeutic agentor agents for treating unwanted cell proliferation.

The agonist can be used in combination with other therapies. Forexample, the combination therapy can include a deacetylation agonist,e.g., a SIR agonist, of the present invention co formulated with, and/orco administered with, one or more additional therapeutic agents, e.g.,one or more anti-cancer agents, hormone treatment, vaccines, and/orother immunotherapies. Such combination therapies may advantageouslyutilize lower dosages of the administered therapeutic agents, thusavoiding possible toxicities or complications associated with thevarious monotherapies. In some embodiments, the administration of adeacetylation agonist enhances, e.g., sensitizes, cancer treatments withother anti-cancer agents. Thus, in some embodiments, the treatment ismore effective because of combined administration. For example, thesecond treatment, e.g., the anti-cancer agent or other therapeuticagent, is more effective, e.g., an equivalent effect is seen with lessof the second treatment, or the second treatment reduces symptoms to agreater extent, than would be seen if the second treatment wereadministered in the absence of the agonist. In some embodiments,delivery is such that the reduction in a symptom, or other parameterrelated to the disorder, is greater than what would be observed with thesecond treatment delivered in the absence of the agonist. The effect ofthe two treatments can be partially additive, wholly additive, orgreater than additive. In some embodiments, the administration of ananti-cancer agent in combination with the agonist, may lower the dose ofthe anti-cancer agent or other therapeutic agents (e.g., hormonetreatments), by at least 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60% or morefrom the dose of the anti-cancer agent or other therapeutic agentadministered in the absence of administration of the agonist.

Administered “in combination”, as used herein, means that two (or more)different treatments are delivered to the subject during the course ofthe subject's affliction with the disorder, e.g., the two or moretreatments are delivered after the subject has been diagnosed with thedisorder and before the disorder has been cured or eliminated. In someembodiments, the delivery of one treatment is still occurring when thedelivery of the second begins, so that there is overlap. This issometimes referred to herein as “simultaneous” or “concurrent delivery.”In other embodiments, the delivery of one treatment ends before thedelivery of the other treatment begins. The delivery can be such that aneffect of the first treatment delivered is still detectable when thesecond is delivered.

In other embodiments, the agonists are administered in combination withother therapeutic treatment modalities, e.g., the existing modalitiesfor treating cancer, including surgery and radiation.

In some embodiments, the agonist can be administered in combination witha high energy radiation emitters, for example, a radioisotope, such as¹³¹I, a γ-emitter. Other suitable radioisotopes include α-emitters, suchas ²¹²Bi, ²¹³Bi, and ²¹¹At, and β-emitters, such as ¹⁸⁶Re and ⁹⁰Y.Moreover, Lu¹¹⁷ may also be used as an anti-cancer agent.

In some embodiments, the second therapeutic agent can be, for example,one or more of a chemotherapeutic agent and a cytotoxin. Examples ofchemotherapeutic agents include taxol, cytochalasin B, gramicidin D,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,busulfan, cisplatin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, chlorambucil, gemcitabine,actinomycin, procaine, tetracaine, lidocaine, propranolol, puromycin,maytansinoids and analogs or homologs thereof. Additional therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, CC-1065, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine, vinblastine, taxol and maytansinoids).

The following examples are merely illustrative of particular aspects ofthe invention described herein.

EXAMPLES

The following examples demonstrate that cytochrome c is deacetylated byhuman SIRT2 and human SIRT3 using recombinant forms of the SIRT proteinsas well as proteins derived from SIRT “pull downs” from cell extracts.The activity of these proteins was determined in vitro using either a¹⁴C-nicotinamide release assay or a titrated acetate release assay.

Example 1 SIRT2 Deacetylation Activity on Cytochrome C

Recombinant GST-SIRT2 deacetylation activity was tested on chemicallyacetylated cytochrome c in the presence and absence of NAD.Deacetylation activity was determined in vitro using a titrated acetaterelease assay.

As shown in FIG. 1, recombinant GST-SIRT2 deacetylates cytochrome c in aNAD dependent manner.

Example 2 Recombinant SIRT3 Deacetylation Activity on Cytochrome C

Recombinant GST-SIRT3 deacetylation activity was tested on chemicallyacetylated cytochrome c in the presence and absence of NAD. Therecombinant SIRT3 was a fragment of human SIRT3 lacking the first 100amino acids of the N-terminus of full length human SIRT3. Deacetylationactivity was determined in vitro using a titrated acetate release assay.

As shown in FIG. 2, recombinant GST-SIRT3 deacetylates cytochrome c in aNAD dependent manner.

Example 3 Cell Derived SIRT3 Deacetylation Activity on Cytochrome C

Cell derived GST-SIRT3 deacetylation activity was tested on chemicallyacetylated cytochrome c. The SIRT3 was derived from “pull down” 293Textracts.

Deacetylation activity was determined in vitro using a ¹⁴C-nicotinamiderelease assay. Deacetylation was determined for GFP (control) withchemically acetylated cytochrome c, SIRT3-GFP with chemically acetylatedcytochrome c, and SIRT3-GFP with histone 4.

As shown in FIG. 3, cell derived SIRT3 deacetylates cytochrome c.

Example 4 Cell Derived SIRT1-7 Deacetylation Activity on Cytochrome C

Deacetylation activity was tested for cell derived SIRT1, Sirt2, SIRT3,SIRT5, SIRT6 and SIRT7 on chemically acetylated cytochrome c. The SIRTenzymes were derived from “pull down” 293T extracts expressing therespective SIRT family member.

Deacetylation activity was determined in vitro using a ¹⁴C-nicotinamiderelease assay. Deacetylation was determined for a control reactionmixture with cytochrome c, SIRT1 with cytochrome c, SIRT2, withcytochrome c, SIRT3 with cytochrome c, SIRT5 with cytochrome c, SIRT6with cytochrome c and SIRT7 with cytochrome c.

The results are shown in FIG. 4.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A method of evaluating a compound, the method comprising contacting apolypeptide having acetylase or deacetylase activity, or fragmentthereof, with a compound, in the presence of a cytochrome c polypeptide,and evaluating if the compound modulates interaction between thepolypeptide and the cytochrome c.
 2. The method of claim 1, wherein thecytochrome c polypeptide is acetylated at at least one lysine.
 3. Themethod of claim 1, wherein the cytochrome c polypeptide is full lengthcytochrome c.
 4. The method of claim 1, wherein the cytochrome cpolypeptide is human.
 5. The method of claim 1, wherein the cytochrome cpolypeptide is a fragment of between 3 and 20 amino acids of a fulllength cytochrome c.
 6. The method of claim 1, wherein the polypeptidehas deacetylase activity and is a SIR polypeptide.
 7. The method ofclaim 6, wherein the SIR polypeptide is a human SIR polypeptide.
 8. Themethod of claim 7, wherein the SIR polypeptide is SIRT1, SIRT2, orSIRT3.
 9. A method comprising: contacting a cell which expresses apolypeptide having acetylation or deacetylation activity and acytochrome c polypeptide with a test compound, and determining if thetest compound modulates acetylation of the cytochrome c polypeptide. 10.The method of claim 9 further comprising evaluating apoptosis or anindication of apoptosis in the cell.
 11. A method of evaluating a testcompound, the method comprising: contacting a polypeptide havingacetylase or deacetylase activity, or fragment thereof, with a testcompound, in the presence of a cytochrome c polypeptide, in vitro, andevaluating if the test compound modulates interaction between thepolypeptide and the cytochrome c; contacting a cell which expresses apolypeptide having acetylation or deacetylation activity and acytochrome c polypeptide with the test compound, and determining if thetest compound modulates acetylation of the cytochrome c polypeptide inthe cell.
 12. A method of evaluating a protein, comprising: identifyingor selecting a candidate protein, wherein the candidate protein is apolypeptide having acetylation or deacetylation activity, or acytochrome c polypeptide; altering the sequence, expression or activityof the candidate protein in a cell or in one or more cells of anorganism; and determining whether the alteration has an effect on theinteraction, of the polypeptide with a cytochrome c polypeptide, or onthe deacetylation of cytochrome c.
 13. A method of evaluating a protein,the method comprising: a) identifying or selecting a candidate protein,wherein the candidate protein is a polypeptide having acetylation ordeacetylation activity, or a cytochrome c polypeptide; b) identifyingone or more polymorphisms in a gene that encodes the candidate protein;and c) assessing correspondence between the presence of one or more ofthe polymorphisms and an interaction of the polypeptide with thecytochrome c, or with the deacetylation of the cytochrome c.
 14. Amethod comprising: providing a cell which expresses cytochrome c andwhich either over- or under-expresses the cytochrome c, contacting thecell with a compound; and evaluating the compound for its ability tomodulate acetylation in the cell.
 15. The method of claim 14, whereincell viability and apoptosis can be evaluated.
 16. The method of claim15, wherein a mitochondrial function is evaluated.
 17. A method ofmodulating cell growth in an animal comprising modulating theacetylation status of a cytochrome c in the animal.
 18. The method ofclaim 17 wherein cell growth is modulated using an antagonist or agonistof a deacetylase.
 19. The method of claim 17 modulating cell growth byincreasing acetylation of cytochrome c.
 20. The method of claim 17modulating cell growth by decreasing acetylation of cytochrome c. 21.The method of claim 18 wherein the deactylase is a human SIRpolypeptide.